Poster demonstrations: The Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, UK, on 19 March, 1981

882

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINH AND HYGIENE, VOL. 75, No. 6, 1981

ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE

Poster Demonstrations

held at

The Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, UK, on 19 March, 1981

Computerized chemical analysis NIGEL BAILEY AND BRIAN CAPEL

$2entre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts, UK

It is well established that infectious diseases frequently induce changes in whole blood and serum components of the infected host. The measurement of such changes may serve to give an early indication of the onset of disease, contribute to understanding of the pathogenesis of infection and may also be of value in both assessing and monitoring prophylactic and therapeutic treat- ments (HAMBLETON, l?. et al., 1978, Brit. J. exp. Path., 59, 630-639).

The host responses to infection may include haematological changes, altered levels of serum electrolytes, trace metals, free amino acids, enzyme activities, various metabolities and also synthesis of acute- phase proteins.

Serum samoles from a susnected infected host can be processed and assayed -for levels of enzyme activities and metabolites. These samples are automatically analysed with a reaction rate analyser and results given by a Kinetic data processor.

Trypanosoma dionisii in “buffalo” lung cells J. R. BAKER,* AUDREY M. GLAUERT AND LINDA F.

SELDEN Molten0 Institute and Strangeways Research Lab- oratory, Cambridge, UK (*Present address: Culture

Centre of Algae and Protozoa, Cambridge). The interaction between Trypanosoma (Schizo-

trypanum) dionisii (see BAKER & SELDEN, 1981, Trans. R. Sot. trop. Med. Hyg., 75, 80-85) and mono- layers of non-phagocytic “buffalo” (bison) lung (BL) cells cultured in vitro for up to seven days has been examined by light and electron microscopy. Epimastigotes of T. dionisii did not appear to enter BL cells. Trypomastigotes entered rapidly, as early as two hours after addition to the monolayer; entry was inhibited by pre-heating (60°C 5 min) the parasites, suggesting an active role for stage- specific, heat-labile surface membrane components in the process. Entry was also inhibited by cyto- chalasin D, which had no effect on the motility of the protozoa, indicating that a normally functioning microfilament system in the BL cells is required for uptake. It was concluded that entry is a process of ‘induced phagocytosis’ in which both parasite and host cell play an active role. Electron micro-

scopy showed that the entry of trypomastigotes into BL cells was unusual in that no enveloping pseudo- podia were produced by the host cell; the parasites appeared to induce the formation of dips or furrows in the cell surface and then to ‘sink into’ the BL cell.

The development of trypomastigotes of T. dionisii within BL cells parallelled that of T. cruzi within macrophages. Shortly after entry the phago- some membrane disintegrated and the free intra- cellular parasites changed into rounded amastigotes one to two days after entry. These amastigotes replicated and then developed into elongated promastigotes and subsequently trypomastigotes. A characteristic feature of the promastigotes was a broad pseudopod which developed from the rim of the flagellar pocket and appeared to be a device for anchoring the parasite in the host cell cytoplasm during the process of elongation. A cytostome was frequently seen at this stage of development. After seven days, intra- and extra-cellular trypomastigotes were present and many BL cells showed signs of lysis and had breaks in the plasma mem- branes, through which the trypanosomes had presumably emerged.

Suspension culture of P. falciparum-manual and semi-automated methods

G. A. BUTCHER C.A.M.R., Porton, Salisbury, U.K. and Guy’s

Hosaital Medical School, London The requirement for fresh human serum may

limit the usefulness of the Trager-Jensen method for large volume Plasmodium falciparum culture. Outdated plasma from transfusion centres is sometimes more readily available but does not give satisfactory in vitro growth in static cultures (JENSEN, 1979, Bull WHO, 57 (Suppl. l), 27; BUTCHER, 1981, Ann. trop. Med. Parasit., 75, 7). However, P. falciparum can be produced in quantity using this serum source in agitated cultures by the simple expedient of shaking the gassed culture flasks in an orbital incubator. Medium, sera, cells, gas, etc., are as previously described (BUTCHER, 1979, Bull. WHO, 57 (Suppl. I), 17; BUTCHER, 1981). Some loss of erythrocytes occurs possibly due to the hypotonicity of particular batches of RPM1 (WEY- MOUTH, 1970, Itt Vitro, 6, 109) or the use of outdated cells. The demonstration showed the growth of parasites in a few representative cultures (50ml

PHLS CENTRE FOR APPLIED MICROBIOLOGY AND RESEARCH, PORTON DOWN, WILTSHIRE 883

suspension per flask). The malaria produced was processed for antigen after initial washes by saponin lysis (l/10,000 Saponin/PBS, for 10 minutes, 4°C) followed by a low speed spin to remove precipitated serum protein etc., and a high speed one to sed- iment parasite material.

A simple semi-automated apparatus was also demonstrated for maintaining cultures (BUTCHER, 1981, Ann. trop. Med. Par&., 75, 111); It con- sists of a 400 ml conical flask containine 50 ml of culture in an orbital incubator (100 rpm).O Medium, gas, etc., are supplied through silicone tubing. Medium is replaced daily (when the shaker stops allowing cells to settle out), by peristaltic pumps under control of a cam-timer.

A solid phase immune cell adherence assay A. C. CH~NAS, E. A. GOULD AND M. G. R. VARM~ Arbovirus Unit. Winches Farm Field Station. 395

Hatfield, 6. Albans, Herts AL4 OXQ, UK: A solid phase test employing a continuous line of

macrophage-like cells of murine origin as a marker for virus antibody-antigen interaction was demonstrated. It measured virus antigens which had been adsorbed to a solid phase either by selection with specific monoclonal antibody or saturation with high concentrations of semi-purified antigen, In this respect the principle resembles tests such as ELISA and RIA but differs from them inasmuch as it employs cultured cells with Fc receptors as markers.

Two-fold dilutions of antibody were added to a flat-bottomed microtitre plate which had previously been pre-coated with antigen. After incubation for one hour at 37°C the plate was washed and a suspension of macrophages in PBS (approximately lo5 cells) was added to each well. After a further one hour incubation at room temperature the plate was washed by total immersion in PBS. Remaining adhering cells were visualized by staining with naphthalene black solution. Antibody titres could then be read visually or after cell solubilization with 0 * 1 y0 Nonidet in a micro-ELISA reader at 620 nm.

In a modification of the above technique specific monoclonal antibodies were used as the first step reagent. This allowed highly specific selective adsorption of antigens from heterogeneous mixtures. Subsequent titration of a Sindbis virus hyperim- mune serum on a plate, thus adsorbed, demonstrated quantitative differences of different antibody levels in the serum. In this case a crude antigen derived from disrupted infected cells was used and monoclonal antibodies to the haemagglutinin (HA), haemolysin (HL), and capsid protein (NP), were used as the first step.

The API as an economic statistic G. E. CUMPER

Evaluation and Planning Centre, Ross Institute, London School of Hygiene and Tropical Medicine,

London, WClE 7HT Economic evaluation of malaria control projects

requires some measure of malaria incidence or

prevalence, so that one can link changes in this measure with: resources devoted to control (cost- efficiency) ; economic effects of control, e.g., reduction in lost working time (benefits). From the statistics routinely collected in malaria programmes, the API seems the best over-all indicator of the national situation. Can we use it for economic evaluation? The following example from Thailand suggests we cannot.

Resources available for the malaria control nroaramme in Thailand fell sharolv between 1969 &d-1974. The crude API rose*sharplv between 1972 and 1974, and more slowly from 1974 to 1978. On this basis it was araued that the situation was running out of control and that resources should be restored to the pre-1974 level. But closer analysis casts doubt on this.

Case detection in Thailand is in three main forms -active (ACD), passive (PCD) and other mis- cellaneous sources (0). PCD yields much higher slide positivity rates than other forms and increased in importance between 1972 and 1978 because of growth in the numbers of primary health centres and malaria clinics. With a nearly constant annual blood examination rate, other, low-yielding forms of case detection declined. This biases the crude API as a measure of population incidence.

Can one adjust the crude API to allow for this? One possibility is to take advantage of the observed linear relationship between the cumulative SPR (SPR,) and the ratio of population to cumulative number of examinations (P/E,) to interpolate/ extrapolate the effect of hypothetical increases in the number of examinations. As P/Et approaches zero the picture becomes one of moderate-increase in incidence between 1972 and 1974. with a steadv movement back toward 1972 levels’ from 1974 m 1978. This suggests that other factors such as increased management efficiency have successfully compensated for the reduction in programme resources.

Research is required to test whether a firm basis can be found (e.g. in mathematical modelling) for some such adjustment of the API for economic purposes. If so, quite simple and cheap surveys may be feasible to link malaria incidence with related economic variables. If not, the problem may call for much more expensive surveys designed expres- sly to monitor the disease and its effects, independ- ent of the statistics currently used to manage malaria control programmes.

The use of plasmid RP4 in genetic studies of Vibrio cholerae 1621

D.A. BROADBENTAND D.&YOUNG London School of Hygiene and Tropical Medicine,

Keppel St., London WClE 7HT Bacterial plasmids of the P-incompatibility

group have a wide host range among Gram negative bacteria (DATTA et, al., 1971, J. Bact., 108, 124). Certain of them, for example RP4, are capable of mobilizing the host chromosome (HOLLOWAY, 1979, Plasmid, 2, 1). RP4 carries resistance deter- minants for tetracycline and kanamycin and also contains the transposable element Tn 1 which

884 POSTER DEMONSTRATIONS

specifies resistance to ampicillin. We have investigated the use of this plasmid in genetic studies of V. cholerae.

The RP4 plasmid was initially transferred from auxotrophic E. coli K12 to V. cholerae 1621 by conjugal transfer. Tetracycline-resistant V. cholerae transconjugants were shown to contain the plasmid RF4 by agarose gel electrophoresis of purified plasmid preparations. RP4 was shown to mediate the transfer of the following chromosomal markers : tryptophan, histidine, adenine, cysteine, lysine, vitamin Bl, serine and arginine. Transfer from V. choZerae strains was affected by incubation at 30°C for five hours.

An RP4 plasmid, temperature sensitive for rep- lication at 42”C, was isolated by HARAYAMA et al., 1980; Mol. gen. Gen., 180, 47). This plasmid was used to insert the Tnl transposon into the V. cholerae chromosome by temperature shift to 42°C followed by selection for ampicillin resistance. Insertion of Tnl from RP4 has been shown to generate auxotrophic mutants of- V. cholerae similar to those -described for S. tryphimurium (KLECKNER et al.. 7. mol. Biol., 116, 125). Also bv reintroduction of is RI’4 into- V. iholerhe carry& chromosomally located Tnl it has been possible to integrate the entire ts RI’4 olasmid into the chromo- som:. Integration is facilitated by the homologous Tnl sequences (KLECKNER et al., 1977). The latter strains are being investigated- for their Hfr potential.

The isolation of Legionella pneumophila from environmental water samples

P.J.DENNIsJ. A. TAYLORAND G.1: BARROW Environmental Hygiene Reference Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK.

The techniques used in this laboratory for the isolation of Legionella pneumophila from environmental water samples were demonstrated. Volumes of 25 litres are filtered through a 142 mm membrane filter of uore ‘size 0 * 2~. The method devised in this laboratoiy uses a peristaltic pump which is faster and safer than pressure filtration. An added benefit is the ability to circulate boiling water through the whole apparatus, achieving adequate disinfec- tion in 5 mins, with a total time between filtration cycles of 10 to 15 min, thus allowing an increased number of samples to be handled with ease.

After filtration of the water, the membrane is macerated in about 30 ml of filtrate, and 5 to 10 ml of the supernatant injected intraperitoneally into each of two guinea-pigs. A sample of the filtrate is analysed for metal and salt ions. The remainder of the macerated membrane is kept, at room temperature, for reference purposes.

The guinea-pig may die as a result of infection; if not, it is killed after 10 days or immediately if a fever develops. A sample of heart blood is taken for measurement of serum antibody levels by indirect immunofluorescence microscopy; the cut surfaces of the spleen and lung are used-to prepare smears and for inculation of Legionella Blood Aaar and Charcoal Yeast Extract ag& plates, modiged

in this laboratory, together with ordinary blood agar as a ‘no growth’ control plate. Isolates are confirmed by gas liquid chromatography and serologically typed by immunofluorescence microscopy.

Enzyme polymorphism in Onchocerca volvulus H.A. FLOCKHARTANDALKARAM

London School of Hygiene & Tropical Medicine, Winches Farm, St. Albans, Herts AL4 OXQ, UK;

OCP, Ouagadougot+ Upper Volta The disease onchocerciasis has long been separ-

ated into two forms, forest and Savannah, the latter causing the more severe eye pathology and blindness DUKE (1976, Tropenmed. Parasit., 27 (Suppl. l), 21-22) has described six strains of 0. voIvulus; these were separated by geographical location, clinical manifestations and vectors.

As these worms appear to be morphologically identical, attempts are-now being made to character- ize the different “strains” bv studvinn their isoenzyme patterns. Parallel investigations with other organisms (trypanosomes, leishmanias and amoe- bae) have shown that in order to determine the extent of variation in each enzyme system used, and hence the relative value of each in subspecific characterization, it is essential to collect a- large number of samnles from several localities. To this end a field trip-was organized to, and paid for by, the Onchocerciasis Control Programme in West Africa. Nodulectomies were carried out in villages in the Savannah and forest areas; the adult male and female worms were removed from the nodules by collagenase digestion, frozen in liquid nitrogen and brought back to England for the isoenzyme work to be carried out.

The preliminary results reported here were obtained from 120 samples of male and female worms examined for four enzymes, lactate dehydrogenase (LHD), malate dehydrogenase (MDH), phosphoglucomutase (PGM) and glucose-phosphate isomerase (GPI). They have shown that multiple forms of LDH and three zymodemes of PGM and MDH exist. All the samples had the same pattern for GPI. Many more enzyme systems remain to be studied but these results do indicate that enzyme polymorphism is occuring in 0. VOZVUZUS and that it may well prove to be a method of separating the various strains.

This work was supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases and by a grant from OCP.

Three aspects of disseminated intravascular coaaulation in subtertian malaria

H. B. GOODALL Ninewells Hospital and Medical School, Dundee,

Scotland, UK This demonstration is ‘based on 10 patients.

Five had no complications and no laboratory signs of disseminated intravascular coagulation (D.I.C.); one had cerebral malaria with transient D.I.C.-


thrombocytopenia, raised fibrin(ogen) degradation products and slightly prolonged one-stage prothrom- bin time; four died and post-mortem tissues from three showed varying degrees of intracapillary thrombosis and sludging of parasitized erythrocytes, but only a thick film was available from the fourth.

The first aspect is the presence of giant nuclear masses in the lungs of three fatal cases and blood from one of these (GOODALL, H. B., 1978, Lancet, ii, 1124-6). While some masses may be megakaryo- cytic, the) are more probably dercved fro& enho- thelium shed in relation to D.I.C. Nuclear masses in blood (huffy coat films) numbered approx. 200 per ml. in one fatal case, 10 in the non-fatal cerebral case and 3 or less in each uncomplicated case.

A mystery of subtertian malaria (second aspect) is how particular capillaries, especially cerebral, are so filled with sludged, parasitized erythrocytes. One fatal case was peculiar in showing involvement predominantly of mid-brain and related to vascular lesions of Wernicke-Korsakov psychosis-a drunk- en Scats engineer aged 52. He also had capillary damage of the colon and ulceration and parasites in erythrocytes in faeces!

Ulceration led to (third aspect) Escherichia coli and Clostridium welchii septicaemia which over- whelmed the patient presumably because of reti- culoendothelial blockade by red-cell debris, parasites and pigment. The final fatal case (a Gambian woman of 20) showed pigment-laden macrophages, scanty parasites, but large bacilli in the thick film. Did she die of septicaemia? Perhaps septicaemia is a more frequent fatal complication than has previously been imagined.

Thanks are expressed to Drs. I. H. Graham, J. Boyd, A. C. Sott, D. MacLean, D. Emslie-Smith, W. M. Jamieson, Mary Kerry, A. J. P. Crowden and N. R. Chahine.

The use of phenylalanine ammonia lyase in the treatment of phenylketonuria G. W. JACK AND H. J. GILBERT

PHLSICAMR, -Therapeutic l%oducts Laboratory Porton Down. Salisburv. Wilts SP4 07G. UK Phenylalanine ammoG;a-lyase (E.c h.3.1.5.)

(PAL) is an enzyme found in plants and fungi which converts I-phenylalanine td trans-cinnamic acid and ammonia (KOUKOL. 1. & CORN. E. E.. 1961. 7. biol. Chem., 236, 2692-2598; HODGI&S, D.‘S., 19?j, J. biol. Chem., 246, 2977-2985). The genetically determined metabolic disease phenylketonuria (SCRIVER, C. R. & CHOW, C. L., 1980, New Engl. J. Med., 303, 1336-1342 & 1394-1400), is currently treated by a synthetic low-phenylalanine diet which is unpalatable and imposes social stress on the patient and family. Enzymic treatment with PAL envisages the patient eating a normal diet supple- mented with PAL in enterically coated capsules to protect its passage through the stomach before release in the small intestine where it can degrade the phenvlalanine release from dietary protein.

The enzyme is partially purified fro& a yeast, Rhodotorula glutinis, then freeze-dried, encapsulated and enterically coated. Initial results (HOSKINS

et al., 1980, Lancet, i, 392-394) suggest that the enzyme has some effect in lowering the rise in circulating phenylalanine following a protein meal in normal individuals and in lowering the circulating phenylalanine in institutionalized phenylketonurics eating a normal diet.

Experiments in vitro with duodenal juice show PAL to have a short half-life in the juice and an extensive programme of chemical modification of the enzyme failed to improve its stability/activity in the juice. The effectiveness of the enzyme in vivo does not correlate with its instability in vitro, suggesting that some degree of compartmentalizat- ion may occur in the small intestine placing the enzyme in a more protected environment. Further work is directed at improving the performance of PAL in viva by presenting the enzyme in a more stable form.

The influence of growth rate and nutrient limitation on the bacterial composition and metabolic activity of a mixed culture of oral

bacteria grown in a chemostat P. D. HUNTERS, J. R. HUNTERS, G. H. BOWDEN~, I. R. HAMILTON%, J. M. HARDIER AND D. C.

ELLWOOD~ ICentre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts., UK; 2Dept. of Oral Biology, University of Manitoba, Winnipeg, Canada; 30ral Microbiology, The London Hospital Medical

College, Turner St., London El ZAD Dental plaque is the microbial community found

on the tooth surface which is embedded in polymers of salivary and bacterial origin. This community lives in harmony with the host but is also associated with the most prevalent diseases currently affecting industrialized societies : caries and periodontal disease. In an attempt to recognize the parameters that may influence the transition of the flora from a comm&sal to a pathogenic relationship with the host. the effect of different growth conditions on the *bacterial composition aid metabolic activity of a mixed culture of oral bacteria was studied. Human dental plaque was grown anaerobically in a chemostat under glucose-limitation and under glucose-excess conditions (pH6.5, 37%). The dilution rate, D, was increased stepwise from 0*05h-l to 0.6h-l (mean community generation time range of 14h to 102h) and then returned to D = 0 * 05h-I. Samples for differential colony counts on selective and non-selective media and for bio- chemical analyses were taken during steady-state conditions at each dilution rate. Contrary to chemostat theory, microbial communities with a high species diversity were maintained under all conditions of growth suggesting that the component populations were participating in a variety of complex inter-relationships. However, the persistence and degree of prominence of bacterial populations within the community, together with the metabolic activity and the microbial composition of the community varied with the growth-limiting nutrient and with the dilution rate. The glucose-limited culture had a more complex flora which produced a wide range of volatile fatty acids as fermentation products,


suggesting amino acid catabolism. Lactate was the major fermentation product of the glucose- excess culture (amino acid limited) which also supported higher numbers of extracellular polysacchar- ide-producing streptococci. Not all of the bacterial populations of the inoculum established successfully in the chemostat.

Isoenzyme characterization of trypanosomes from fish and birds

CAROL A. LETCH AND JIRI LOM London School of Hygiene and Tropical Medicine London WCEl 7HT and Czechoslovak Academy of

Sciences, Prague, Czechoslovakia Trypanosomes isolated from the blood of fresh-

water carp, perch, bream, tenth and pike from rivers in Czechoslovakia, and a stoneloach from the River Lee in Hertfordshire, UK, could be classified into 3 groups on the basis of the computed similarity indices of the zymodemes of 16 enzymes, as visualized on starch gels. The groupings had no relation to the species of fish from which the stocks were isolated and LETCH (1979, Parasitology, 79,107-l 17) had previously demonstrated that trypanosome infections in 3 fish species from the River Lee were identical for 11 enzymes. The results indicate that the fish trypanosomes cannot be classified as separ- ate species but merely a series of stocks able to infect a wide variety of fish. The absence of host specific- itv as revorted bv LOM (1973. ‘Y. Protozool.. 20. 537) and‘LETCH (1979) supportstithis view. ’ ’

A similar isoenzyme study was carried out with 9 bird trypanosomes, eight isolated from the blood of blackbirds, chaffinches, greenfinches and rooks from Czechoslovakia and one from a rook in Hert- fordshire. The nine stocks could be classified into five groups on the basis of their isoenzyme patterns but three of the groups were fairly similar. The other 2 groups consisted of trypanosomes from the blood of a Czechoslovakian rook and a rook from Hertfordshire. which not onlv d&fered from each other but also from the other-groups. One of the blackbird stocks was enzymatically identical to another from the blood of a greenfinch, both samp- ples originating from the same area but collected three years apart. These results suggest that, as with the fish-trypanosomes, there is% general a loose host specificity, stocks being capable of infecting a wide variety of bird species.

Canine leishmaniasis-autopsy report of an imported case

J. A. LONGSTAFFE, M. W. GUY* AND P. G. C. BEDFORD

The Royal Veterinary College Field Station, Hawks- head Lane, North Mymms, Hatfield, Herts, UK; *London School of Hygiene and Tropical Medicine,

London WClE 7HT History: The subject, an eight-year-old spayed female Afghan hound, spent two years in Athens before re-importation to the UK in 1977. Visceral

leishmaniasis was diagnosed just before re-importation and a course ofNa stibogluconate was given.

In 1978, following episodes of conjunctivitis and rhinitis the dog was admitted to hospital where smears, IFA and skin tests were negative for leishmaniasis. However. further treatment with Na stibogluconate was carried out and apparent com- plete recovery was affected.

The animal was re-presented in December 1980 with a history of wasting, diarrhoea and ulcerative lesions of eyelids, lips and nasal mucous mem- branes. Smears from lesions were negative for amastigotes. Euthanasia was performed in January 1981.

Gross Autopsy Findings: The carcass was emaci- ated with slight hair loss, there was marked kerato- conjunctivitis of one eye, no skin ulcers were observed. The spleen and liver were enlarged as were all lymph nodes which on incision showed a thin rim of cortical tissue surrounding an enlarged brown medulla. Histological Findings : Multiple focal granulomatous lesions consisting of macrophages (containing amastigotes) and plasma cells were found in the dermis, cornea, conjunctiva, iris and ciliary body, adrenals, liver, pancreas, intestinal mucosa, renal pelvis, lung and myocardium. Bone marrow, spleen and lymph nodes contained very large numbers of parasitized macrophages and plasma cells. Discussion : This is the third case of imported canine visceral leishmaniasis diagnosed in the UK. It is intended that a fuller account of all three will be published to draw attention of UK veterinary practitioners to a potentially zoonotic disease that can appear in imported dogs long after the quaran- tine period is over.

A rapid method for the infec‘tion of mice with Schistosoma japonicum

N. A. MOLONEY AND G. WEBBE Dent. of Medical Helmintholoev. London School of Higiene and Tropical Medicin; 395 Hatfield Road,

St. Albans, Herts AL4 OXQ, UK Methods which have previously been used for

infecting laboratory animals with Schistosoma japonicum are tedious, unsafe or inaccurate (MELENEY et al., 1953; Amer. J. trop. Med. Hyg., 2, 883-913 SADUN & WALTON, 1958; AmerJ. trop. Med. Hyg., 7, 500-504). This presentation indicates that they might be superseded by an injection method of infection. Cercariae were suspended in medium and known numbers were injected intraperitoneally or subcutaneously into mice and were compared for infectivity with S. japonicum cercariae that had been administered percutaneously bv the bacterial loop/coverslip method (Hsu et al.,-1965; 2. Tro- aenmed. Parasit.. 16. 83-89). Iniected cercariae were no less infective or fecund than those percutaneously applied. The intraperitoneal route of infection was preferred to the subcutaneous one because of the more consistent worm yields produced. Juvenile worm burdens and mean worm sizes were compared and found to be no different in mice receiving cercariae intraperitoneally or percutaneously. Worm


yield was not impaired by in vitro incubation of cercariae in medium before injection. This new method of infecting animals with S. japonicum also has advantages over previous techniques in terms of time and safety.

Reliability of superoxide dismutase isoenzyme for distinguishing A. merus from other member

of the Anopheles gambiae complex L. M. OKETCH AND G. B. WHITE

International Centre for Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya; Dept. of Entomology, London School of Hygiene and Tropical Medicine, London WClE 7HT

Among six sibling species of mosquitoes com- prising the A. gambiae complex, diagnostic iso- enzymes have been reported in samples of A. quadriannulatus from southern Africa, species D from Uganda, A. melas from West Africa and A. merus from eastern Africa (MAHON et al., 1976; Bull. ent. Res., 66, 25-31; MILES, 1978; Bull. ent. Res., 68, 85-96). A particular slow electromorph of superoxide dismutase (tetrazolium oxidase) in A. merus serves to distinguish the adult females of this saltwater-tolerant species from those of the three freshwater-breeding species A. arabiensis, A. gambiae and A. quadrinnulatus, all of which have an electromorph with faster mobility during starch gel electrophoresis, as shown in this demonstration.

In preparation for studies on the distribution, ecology and vector functions of A. merus in Kenya, we have ascertained that the specific phenotype of superoxide dismutase can be detected in males as well as females, and in mature larvae (stages II- IV) and pupae reared from eggs obtained from female mosquitoes collected on human bait at Vanga, Kenya, in Febuary and March 1981. Since the diagnostic isoenzyme was present in putative A. merus larvae, pupae and adults reared in London tapwater, we consider that the distinctive electromorph is not induced by salinity, and we confirm that it appears to be a constant characteristic, reliable for identification of A. merus adults of both sexes and immature stages from freshwater or saltwater breeding sites.

Host-range and host-parasite relationships of the nematode Neoaplectana carpocapsae and

some arthropods of medical importance W.J.C. OSWALD AND D.M. MINTER

Dept. of Entomology, London School of Hygiene and Tropical Medicine, Keppel Street, London

WClE 7HT The nematode Neoaplectana carpocapsae is a

lethal obligate parasite of terrestrial insects. Death of the host results from a septicaemia caused by a bacterial svmbiont (Xenorhabdus nematonhilus), which is released into the haemocoel. Two fuii generations of the worm usually develop in the cadaver, but new infective stages are eventually produced as food resources are depleted; these then actively leave to seek new hosts.

The susceptibility of adults and immature stages of 16 arthropod species was tested in the laboratory with a range of doses. The over-all pattern of susceptibility was : Heteroptera > Siphonaptera > Diptera > Orthoptera. Some dip- terous larvae and adults were very susceptible, but not pupae; nymphs of Heteroptera were moderat- ely susceptible, but the tick Rhipicephalus appen- diculatus was totally resistant.

Triatomine bugs are readily infected and mor- tality rates at low doses are high (76 to 100%); usuallv there is urolific develoument of new infective stages. However, the presence of fresh blood in the gut of triatomines can have an inhibitory effect on the host-parasite relationship. In the cadavers of bugs killed by a dose of 20 nematodes/ insect, nematode development ceased at the second generation of 10% and 18% respectively of recently fed adult Rhodnius prolixus and Triatoma infestans, whilst development was arrested in only 5% and 4% of unfed adults of the same two species.

Exposure of adult T. prolixus and T. infestans to a low dose (20/insect), although killing the bugs may result in unisexual worm infections and a consequent lack of production of new infective stages by up to 42% of R. prolixus and 70% of T. infestans. However, when fed and unfed insects are killed by this low exposure dose of 20 worms per insect, 65% (R. prolixus) and 55% (T. infestans) will none the less each produce a mean of 5440 (R. prolixus) and 13,360 (T. infestans) new infective stages. This represents a very large net gain in new infectives liberated, and indicates that N. carpocapsae has considerable potential for biological control of triatomines.

Congo-Crimea haemorrhagic fever in Iraq 1979

G. S. PLATTE, T. SOUTHEE~, NICOLA JONES~, E. T. BOWEN~, D. I. H. SIMPSON~, D. S. ELLIS

AND SUSAN STAMFORD~ %entre for Applied Microbiology and Research, Porton Down, Salisbury, Wilts, UK: 2London School of Hygiene and Tropical Medicine, London

WClE 7HT A small outbreak of Congo-Crimea haemorrhagic

fever was recognized for the first time in Iraq/in late 1979. 10 patients were investigated: eight gave a history of contact with sheep or cattle whose Hyaloma ticks are generally associated with viruses of the Congo-Crimea group. The other two patients, a doctor and a nurse, acquired their infections in hospital by direct contact- with a patient.

The illness nresented with severe headache, fever, backache and vomiting, followed by diarrhoea; conjunctivitis and severe bleeding from multiple sites, including gums, nose and urogenital tract. Haematomas were common and large entravasations of blood on limbs caused extensive echymoses. Seven patients died.

Primary isolation of the infective agent was via a patient’s serum into suckling mice. Serum from those mice was used to infect BHK cells, which were then studied by electronmicroscopy. After nine days’ infection, some viral cores were seen budding

,


into vacuoles. Though usually found singly, up to three particles could be found within a single vacuole. The virions were 85 to 95 nm in diameter and surrounded by a unit membrane. The cores contained electron-dense elements, rather similar to those seen in some arenaviridae.

Negatively stained particles were very fragile. Their over-all diameter was 120 to 130 nm, including spikes 15 mn long. These spikes appeared as hollow tubes. and usuallv numbered 20 around the particle’s equator (S~LEIMAN et al., 1980, Lancet, ii, 931-941).

Embryogenesis of Hyalomma rufipes rufipes Koch, 1844

MD. H. RAHMAN Dept. of Veterinary Parasitology, Liverpool School

of Tropical Medicine, Liverpool L3 5QA The development of the cattle tick Hyalomma

rufipes rufipes was studied from oviposition to hatch. Previous information on the early stages of development in ticks was scanty (AESCH~JMA<N, A. 1958, Acta trop., 63, 1248-1256; ANDERSON, D. T. 1973, Embryology and Phylogeny in Annelids and Arthropods. Oxford: Pergamon Press).

Eggs were collected from Day 0 to 38 (hatch) and incubated at 26 i 1°C and 85% R.H. Whole mounts were urenared in liauid oaraffin for studv of the external features; for hisiology, eggs were fixed for 24 hours in cold acetone, then passed through chloroform, chloroform + paraffin and paraffin before blocking and cutting at 5 to 7~ thickness. Sections were stained with haematoxylin/ eosin and mounted in D.P.X.

The ovoid eggs average 537 i 9.9 x 18.6~. The chorion is thick, elastic and smooth, and the vitelline membrane is thin and transparent. Be- neath the vitalline membrane is a thin layer of peri- plasm, which overlies yolk globules of various sizes suspended in cytoplasmic reticulum. The nucleus is centrally located in a cytoplasmic hollow.

Cleavage begins within 12 hours of ovioosition: at first th&e is a high degree of synchrony in division but this becomes less obvious as cleavage progresses. Cleavage results in an increase in nuclei and an outward movement of nuclei: all cleavage nuclei migrate to the periphery, forming the syncytial blastoderm. The cellularization of the blastoderm leads to the formation of greatly flattened blastoderm cells which later become columnar. Soon the cellular blastoderm shows anterior and posterior regions with advanced mitotic stages: some cells migrate back to the yolk as vitellocytes. The germin- al primordium is formed by mitotic divisions and cellular aggregation by contraction. Bilateral thick- ening of the germ band next occurs and gastrulation is by scattered migration of cells beneath the surface to form an inner mesodermic layer. When the bilateral germ band occupies nearly two-thirds of the egg circumference, segmentation becomes evident. Almost coincidently with the segmentation of the ectodermic layer (as outgrowth of future appendages) the division of the mesodermic layer into corresponding somites occurs by ‘rolling in’. The splanchnic layer of the mesoderm gives rise

to dorso-ventral muscles. The vitellocytes participate in the formation of midgut epithelium and malpighian tubules. The nervous system primordia are a pair of ectodermal thickenings in each append- age segment and opisthosomal segment. Later these segments condense round the oesophagus to form the supra- and sub-oesophageal ganglia, usually after the longitudinal contraction of the two halves of the germ band.

A fourth pair of limb buds appear but these remain rudimentary, disappearing before the hatch. When the larva is fully developed, the legs begin to move and these participate in the rupture of the egg-shell. Thelarva pushes itself out of the egg-shell backwards on about Day 38.

Observations on Schistosoma leiperi V.R. SOUTHGATE, R.J. KNOWLES AND G. C.Ross Dept. of Zoology, British Museum (Natural History), Cromwell Road, South Kensington, London SW7 5BD

Schistosoma -leiperi, a parasite of herbivores, was first described bv LE Roux (1955: Trans. R. Sot. trop. Med. Hyg.,.49, 293-294). S. leiperi is found in parts of Botswana, Eastern Caprivi, Zambia and Tanzania. Little further was published about S. leiperi until PITCHFORD (1975, Trans. R. Sot. trop. Med. Hyg., 69, 362) succeeded in introducing it to the laboratory. However, there is still a paucity of information on S. leiperi and a number of techniques have been employed to build up basic information on this parasite.

SEM’s of S. leiperi from Zambia reveal individual variation in the morphology of the tubercles, gar- titularly regarding the degree of spination. S. leiperi from Botswana and Zambia develop well in snails belonging to the Bulinus africanus group (B. africanus. B. alobosus, B. hightoni and B. nasutus) and B. reticu‘iatus group (B wrighti), but are in: compatible with snails belonging to the B. tropicus/ truncatus and B. for&ah group.

The growth and maturation of S. leiperi from Botswana was recorded from 40 to 100 days post- infection in hamsters. The cross-over point (that point at which males and females are the same length) occurs at 44 days p.i. At 40 days p-i. no eggs are seen in the uteri of the females, but at 50 days p.i. 86.3% of the females are gravid. The prepatent period in hamsters is 49 days.

Preliminary studies on chromosomes show that S. leiperi has a complement of 2n = 16.

Phosphoglucomutase, glucose-&phosphate dehydrogenase, acid phosphatase, glucose phosphate isomerase, malate dehydrogenase, lactate dehydrogenase and hexokinase enzymes of adult worm have been studied by isoelectric focusing so that interspecific comparisons can be made.

Is there specific mate recognition in schisto- somes?

V. R. SOUTHGATE. D. ROLLINSON, G. C. Ross AND k. J. KNOWLES-

Dept. of Zoology, British Museum (Natural History), Cromwell Road, London SW7 5BD

[To be published in Parasitology, 1981.1


A non-pathogenic Vibrio for the routine quality control of TCBS medium

J. A. TAYLOR AND G. I. BARROW Environmental Hygiene Reference Laboratory, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, UK

It is important that selective media used for the isolation of bacterial pathogens should be consistently reliable and, considering the potential hazards of laboratory-acquired infection, it is ideal to use a non-pathogenic indicator organism for routine quality control purposes. Such a strain to replace Vibrio cholerae in the quality control of thiosulphate citrate bile salt sucrose (TCBS) medium (TAYLOR, J. A. &BARROW, G. I., 1981, J. clin. Path,, 34,208- 212) was sought among a large collection of freeze- dried vibrios isolated mostly from environmental sources.

A strain was selected which, while closely following the pattern of sensitivity of reference strains and recent isolates of V. cholerae and V. aara- haemolyticus, was consistently more sensit&e to inhibition of growth. This strain was also selected because of its ability to survive in alkaline peptone water and to produce acid from sucrose so that it could be used to demonstrate cholera enrichment and isolation techniques and yield yellow colonies on TCBS medium, like V. cholerae. It can be differentiated from V. cholerae by its salt requirement for growth, the formation of gas as well as acid from glucose and the production of arginine dihydrolasebut not lysine or ornithine decarboxyl- ase. It was identified as a member of Vibrio SD. Group F biotype 2, which has now been named as a new species Vibrio fluvialis (LEE et al., 1981, J. appl. Bact., 50,73-94). The aerogenic biotype 2 is regarded as an environmental organism with no k&wn pathogenic role.

This strain is available from the National Coll- ection of Type Cultures as NCTC 11218.

Transplantation of Onchocerca into mice SIMONTOWNSON, K. EL SINNARYAND A.E. BIANCO London School of Hygiene and Tropical Medicine, Winches Farm Field Station, 395 Hatfield Road,

St. Albans, Herts, AL4 OXQ, UK. Adult males of Onchocerca gutturosa were dis-

sected out of the connective tissue of the ligamen- turn nuchae of naturally infected cattle. Rodents were anaesthetized and the worms introduced into the peritoneal cavity through a small incision which was subsequently closed with sutures. Most worms were recovered from the peritoneal cavity, although occasionally they were found in the thoracic cavity,

From a group of 12 CBA mice of either sex necropsied after 30 days, 15 of the 24 worms trans- nlanted were recovered; of these, 13 were alive and active although some were partially encapsulated or had adhering cells. From a groun of eight CBA

- . v ~~~

mice necropsied after 90 days, eight worms were recovered out of 16 transplanted of which three were alive. All of these worms were partially or wholly encapsulated; some were partially calcified and others were in an advanced state of degenera- tion. In further experiments male worms lived up

to 104 days in the peritoneal cavity of normal CBA mice and up to 133 days in T-cell deprived mice (thymectomized and treated with rabbit anti-mouse thymocyte serum).

However. motile adult female worms of 0. gutturosa that had been digested free of cow tissue with collagenase enzyme were rapidly killed when transplanted into T-cell deprived mice. Female worms transplanted within bovine connective tissue into T-cell deprived mice lived up to 71 days and produced microfilariae that migrated to the ears.

Male and female worms transplanted in bovine connective tissue lived up to seven days in the peritoneal cavity of jirds and guinea-pigs and up to 20 days in Sprague-Dawley rats when microfilariae were recovered from earskin.

A histogram illustrated that mice containing transplanted adult male 0. gutturosa were signifi- cantly resistant to a subsequent inoculation of 0. Zienalis microfilariae when compared to controls.

Sporadic acute non-B hepatitis in England E. M. VANDERVELDE AND I?. P. MORTIMER

Public Health Laboratory Service Virus Reference Laboratory, Central Public Health Laboratory,

Colindale Avenue, London NW9 5HT The aim of this study was to establish whether

sporadic cases of hepatitis non-A non-B occurred in England, and, if so, to compare their character- istics with those of cases of hepatitis A.

Sera from cases of acute hepatitis negative by routine tests for HBsAg were tested for IgM antibody to hepatitis A virus. Reports to the clinicians of the results were accompanied by a questionnaire concerning the final diagnosis and associated features

Of the first 407 sera tested 143 were shown by the presence of specific IgM antibody to be from cases of acute hepatitis A. Of the other 264, 121 had no antibody to hepatitis A, and the remainder had antibody but no specific IgM.

176 replies to the questionnaire were received from the cases in which tests for hepatitis A and B were negative and in 86 of these the final diagnosis remained acute viral hepatitis. The epidemiology of these cases were compared with that of the cases of acute hepatitis A.

In both groups of cases half the patients were between 21 and 40 years of age. Most of the remaining patients with hepatitis A were under 21 but of the remaining uatients with henatitis non-A non-B most were o&r-40. There were approximately equal numbers of patients of each sex in each group.

Two thirds of the patients with hepatitis A, but less than half of those with hepatitis non-A non-B gave a history of either recent travel abroad or of contact with another case of hepatitis. Employ- ment in possible at-risk occupations (for instance, hospital work) and a history of recent hospital treatment, with and without transfusions of blood and blood products, were both more common in patients with hepatitis non-A, non-B than with hepatitis A. Prolonged illness more commonly followed hepatitis non-A, non-B than hepatitis A.


Esterase electromorphs as indicators of organophosphate susceptibility status

of Culex quinquefasciatus mosquitoes F. VILLANI AND G. B. WHITE

Dept. of Entomology, London School of Hygiene and Tropical Medicine, London WClE 7HT

Resistance to organophosphate larvicides is increasingly an obstacle to control of Culex quinquefasciatus mosquitoes, and may be correlated with elevated activity of non-specific esterases (CURTIS 81 PASTEUR, 1981; Bull. Ent. Res., in press). We have begun to investigate the occurrence of normal and abnormal esterase electromorphs in relation to this phenomenon.

Typically, the susceptibility of C. quinquefasciatus larvae to chlorpyrifos (dursban) gives LCss = 0.00065, LCs,, = 0*00093 ppm values, as demonstrated for a Sri Lankan strain. Selected OP-resistant populations may have LCss =0 * 034, LCss = 0.103 aa. shown in line 3 for a Tanzanian strain. Intermediate dose-response values have been obtained for various other strains, such as the more sloping line demonstrated for a non-selected populations from Monrovia, Liberia, thought to be polymorphic for at least one factor causing Or-resistance.

When subjected to starch gel electrophoresis, larval and adult specimens of the Sri Lankan strain were found to be polymorphic for a pair of esterase electromorphs of low intensity (L-bands) and medium mobility. The Tanzanian strain showed only one pair of heavily staining esterase electromorphs (H-bands), the fast having equivalent mobility to that in Sri Lanka. This fast H-band appears violet in colour, and the slow H-band brown, using a + p naphthyl acetate substrate and Fast Blue RR stain. The Monrovia strain was found to be polymorphic for presence or absence of paired H- bands equivalent to those in Tanzania strain, plus L-bands, some of which showed equivalent mobility to those from Sri Lanka.

From the Monrovia stock, sub-strains have been selected for resistance and susceptibility to chlorpyrifos. Theformer is monomorphic for H-esterase, the latter polymorphic for L-esterases, although no attempt was made to select for these phenotypes. Independently, sub-strains have also been selected for H-esterase and L-esterase of a particular type; in the F6 the former has LC5s =0 * 011 while the latter has LCs,-, =0*0021. We take this to confirm that H-esterase is asociated with Or-resistance and serves as an indicator of it. We are now studying the inheritance of several apparently monomeric L-esterase electromorphs, so that genetic relationships of H-esterase can be interpreted.

A study of renal pathology due to Schistosoma haematobium in rural communities in

The Gambia H. A. WILKINS, J. H. AMUASI*, J.C.W. CRAWLEY,

A. G. CRONQUIST AND N. VEALL Medical Research Council Laboratories, Fajara, The Gambia; Radioisotopes Division, MRC Clinical

Research Centre, Harrow, Middlesex, UK. Most studies of the pathology of the renal tract

and kidneys due to Schistosoma haematobium

have been based upon radiological studies (intravenous pyelography) or radioisotope renography carried out in major clinical centres. An assessment of the importance of this pathology to rural communities requires measurements that can be carried out in the field in a manner that is acceptable to the population involved. Radioisotope renography is sensitive to ureteric obstruction, relatively simple to perform and involves minimal patient discom- fort. The absorbed radiation dose is small compared with that due to intravenous pyelography, even in patients with impaired renal function.

Renography involves the i.v. injection of about 1 ml of isotonic saline containing radioactive sod- ium iodohippurate (‘Hippuran’). The renal uptake and release of this tracer is monitored for the following 20 min using detectors over the kidneys whilst a third detector, over the head, monitors blood concentration, A single blood sample is taken for an estimate of effective renal plasma flow (ERPF).

A transportable renography apparatus has been built for use in rural areas of The Gambia where patients with S. haematobium infection have been studied for several years. The system fits into six metal boxes which can be sealed against dust and moisture and carried in the back of a Land-Rover. The apparatus operates from rechargeable batteries floating across a small petrol generator or if neces- sary from the Land-Rover engine. A charger can be used to supply power to the apparatus in the laboratory, where the rechargeable batteries give immunity to the fluctuating mains power supply.

A chart recorder is used to give immediate qualitative assessment of the patient’s condition and the results are analysed to give indices of renal function. These include mean renal transit time (MTT) and percentage ERPF to each kidney.

Measurements on subjects in the non-endemic area (where S. haematobium hasnever been observed) are being compared with measurements on patients in the endemic area of The Gambia where there is a very high level of infection.

*On leave from the Ghana Atomic Energy Com- mission, Legon-Accra, Ghana.

Viral infections as causes of pyrexia of un- known origin in travellers from tropical

Africa A. W. WOODRUFF, E. T. BOWEN AND G. S. PLATT Medical Unit, Hospital for Tropical Diseases,

London, NW1 OPE; Microbiological Research Establishment, Porton, Salisbury, Wilts, UK The sera from 86 travellers who had arrived in

Britain from tropical Africa were examined for the presence of antibodies to a wide range of African viruses, 57 in number and including Lassa fever antibodies. All travellers had suffered from pyrexia of undetermined origin while in Africa. One of the questions asked in carrying out this study was whether Lassa antibodies are commonly encountered among travellers from Africa.

No Lassa fever antibodies were encountered but antibodies to chikungunya, O’nyong-nyong, dengue,

PHLS CENTRB FOR APPLIED MICROBIOLOGY AND RESEARCH, PORTON DOWN, WILTSHIRE 891

Ntaya and Zinga viruses were all found. One of the patients who had Ntaya antibodies gave a history of a neurological illness which had probably been caused by this infection.

Poster demonstration of microbiological safety methods A. E. WRIGHT

Microbiological Safety Reference Laboratory, CAMR, Porton Down, Salisbury, Wilts SP4 OJG, UK

The Microbiological Safety Reference Laboratory mounted a display of teaching materials available in the field of safety with particular reference to the Health and Safety at Work Act as it relates to laboratory workers.

Included in the display was equipment designed to reduce hazards in the laboratory and included pipetting devices, loop incinerators, sealed centri- fuge buckets and containers for carrying toxic material. Packaging materials used in transporting infectious agents as laid down in IATA Regulations were also available for inspection.

Methods of fumigation using formalin were shown together with a variety of designs of discard boxes and other containers for disinfectants.

The equipment used for testing microbiological cabinets and HEPA filters was available for demonstration. Included amongst this were slit sam- plers, Collison nebulisers, anemometers, U.V. intensity meters and detectors for formaldehyde.

Methods for the protection of the individual included protective clothing such as the Martindale suit and the personal S6 respirators.

Procedures for medical monitoring and accident reporting were illustrated.

Lipids of the leprosy bacillus DOUGLAS B. YOUNG

Dept. of Medical Microbiology, London School of Hygiene and Tropical Medicine, London WClE 7HT

Thin-layer chromatography has been used to compare lipid extracts from lepromatous leprosy skin biopsies with those from normal skin and from Mycobacterium leprae purified from armadillo spleen. Several lipids were found in infected skin which were absent from normal skin but corres- ponded to lipids present in the purified M. leprae. These included: (1) Mycolic acids. The pattern of mycolic acids in the cell wall of M. leprae differs from that of all other mycobacteria so far inves-/ tigated. (2) A glycolipid mycoside. A glycolipid containing 6-deoxyhexose sugars but no peptide component was found in M. leprae and in leprosy- infected tissues. Preliminary characterization suggests that this lipid is related to the A and B type mycosides associated with M. kansasii and a. bo&. M. leprae extracts contained no C-type mvcosides. (3) Phthiocerol dimvcocerosate (DIM). This lipid, ‘previously thought to be specific to M. tuberculosis and M. bovis is also synthetized by M. leprae.

Quantitative analysis of M. leprae lipids in skin biopsies from lepromatous leprosy patients indicated that their concentrations were much higher than would be predicted from the number of acid-fast bacilli present. Accumulation of lipid debris from dead M. leprae may provide a protective environment in infected ce!ls for remaining viable bacilli.

The leprosy bacillus synthesizes a characteristic pattern of lipid components which allows it to be distinguished from other mycobacteria. Inability to clear mycobacterial lipids from the site of infection may play a role in the pathogenesis of leprosy. (This work was carried out at The Foundation For Medical Research, Bombay, India.)

Documents

Poster demonstrations: The Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, UK, on 19 March, 1981