In the present paper, we have analysed the cellular and extracellular proteolytic activity profiles in 2 distinct Leishmania braziliensis strains: a recently isolated (virulent) and a laboratory-adapted (avirulent) strain. Quantitative and qualitative differences on the peptidase expression were observed in both strains. For instance, low-molecular mass acidic cysteine peptidase activities were detected exclusively in the virulent strain. Similarly, metallopeptidase activities were mainly produced by L. braziliensis virulent promastigotes. Interestingly, metallo- and cysteine peptidase activities were drastically reduced after several in vitro passages of the virulent strain. Western blotting, flow cytometry and fluorescence microscopy analyses were performed to detect homologous of the major leishmania metallopeptidase (gp63) and cysteine peptidase (cpb) in virulent and avirulent strains of L. braziliensis. Our results revealed that the virulent strain produced higher amounts of gp63 and cpb molecules, detected both in the surface and cytoplasm regions, than the avirulent counterpart. Metallo- (1,10-phenanthroline and EGTA) and cysteine peptidase (E-64) inhibitors arrested the growth of L. braziliensis virulent strain in a dose-dependent manner, as well as the association index with peritoneal murine macrophages. Conversely, these peptidase inhibitors did not affect either the proliferation or the cellular interaction of the avirulent strain. Corroborating these findings, the pre-treatment of the virulent strain with both anti-peptidase antibodies promoted a prominent reduction in the interaction with macrophages, while the association index of the avirulent strain to macrophage was only slightly diminished. Moreover, the spent culture medium from virulent strain significantly enhanced the association index between avirulent strain and macrophages, and this effect was reversed by 1,10-phenanthroline. Collectively, the results presented herein suggest that peptidases participate in several crucial processes of L. braziliensis.

For some years past in Northern Province, Nyasaland, work has been in progress to determine the mortality amongst native cattle due to east coast fever (Wilson, 1944). As an integral part of such a survey, routine tick collections were made during cattle inspections. It was then intended to map the various species of tick concerned in the hope that their geographical distribution might offer a partial explanation for the prevalence or absence of east coast fever in certain areas. Such a map for the western portion of Kenya Colony has been provided by Lewis (1939), ‘according to the groups of dominant species of ticks’; but in Kenya, climate, soils and vegetation show wide contrasting variations, and conditions are scarcely comparable to those existing in the smaller territory of Nyasaland.

Sphingolipids (SLs) are an integral part of all eukaryotic cellular membranes. In addition, they have indispensable functions as signalling molecules controlling a myriad of cellular events. Disruption of either the de novo synthesis or the degradation pathways has been shown to have detrimental effects. The earlier identification of selective inhibitors of fungal SL biosynthesis promised potent broad-spectrum anti-fungal agents, which later encouraged testing some of those agents against protozoan parasites. In this review we focus on the key enzymes of the SL de novo biosynthetic pathway in protozoan parasites of the Apicomplexa and Kinetoplastidae, outlining the divergence and interconnection between host and pathogen metabolism. The druggability of the SL biosynthesis is considered, alongside recent technology advances that will enable the dissection and analyses of this pathway in the parasitic protozoa. The future impact of these advances for the development of new therapeutics for both globally threatening and neglected infectious diseases is potentially profound.

Widespread access to the internet is offering new possibilities for data collection in surveillance. We explore, in this study, the possibility of using an electronic tool to monitor occurrence of the tick vector of Lyme disease, Ixodes scapularis. The study aimed to compare the capacity for ticks to be identified in web-based submissions of digital images/photographs, to the traditional specimen-based identification method used by the provincial public health laboratory in Quebec, Canada. Forty-one veterinary clinics participated in the study by submitting digital images of ticks collected from pets via a website for image-based identification by an entomologist. The tick specimens were then sent to the provincial public health laboratory to be identified by the ‘gold standard’ method using a microscope. Of the images submitted online, 74·3% (284/382) were considered of high-enough quality to allow identification. The laboratory identified 382 tick specimens from seven different species, with I. scapularis representing 76% of the total submissions. Of the 284 ticks suitable for image-based species identification, 276 (97·2%) were correctly identified (Kappa statistic of 0·92, Z = 15·46, P < 0·001). This study demonstrates that image-based tick identification may be an accurate and useful method of detecting ticks for surveillance when images are of suitable quality.

The embryonic epithelium in Hymenolepis diminuta appears in the early preoncosphere stage. Inside the embryo there is a binucleate cell connected by a cytoplasmic strand with an epithelial layer spreading over the embryonic surface. After the embryo has become covered by tine epithelium the latter delammates into three layers. A basal layer resting on the basal lamina accumulates dense bodies. These bodies are spheroid and membrane-bound in the early embryo. In the late preoncosphere stage rodlike bodies remain in the basal epithelial layer. The basal membrane forms long invaginations into the basal layer. The intermediate epithelial layer is rich in polysomes and it is postulated that secretes extracellular material which cements the intermediate and peripheral layers and the ‘oncospheral membrane’. The continuity in embryonic, larval and adult cestode epithelium is discussed.

In 1931 Dr M. V. Lebour drew my attention to a larval Trematode (one of the “Hugetailed Monostome” group of H. M. Miller, 1925) which occurred quite frequently in Turritella communis from the Rame Mud, Plymouth. During 1932–4 I examined 541 Turritella communis from the same locality and an 8 per cent. general infection with four species of closely related cercariae was recorded, but the species which was common the previous year did not occur once. 180 Turritella communis from Naples revealed a 5 per cent, infection of two other closely related species.

1. Mytilicola intestindlis (Steuer) was found in 100% of mussels examined from Blyth, Northumberland. These mussels varied in length between 3·5 and 7·5 cm.

2. The degree of infestation varied from two to fifty-nine parasites per mussel.

3. There was an inverse relationship between the condition of mussels of the size group 5·5–5·9 cm. and both the mean number of parasites per mussel and the mean number of parasites over 1·5 mm. in length per mussel.

4. The mean weight of flesh per mussel in the size group 5·5–5·9 cm. (shell length) was 3·206 g. from Blyth (parasitized) and 5·975 g. from Conway (not parasitized).

5. It is concluded that the presence of Mytilicola is associated with a serious reduction of condition in infested mussels.

It has been reported that infection with Nippostrongylus brasiliensis induces villus atrophy with various histological alterations. In N. brasiliensis-infected rats, villus length in the jejunum was reduced significantly at day 10 p.i., when serum levels of rat mast cell protease (RMCP) II had increased significantly. To determine whether the villus atrophy is associated with enhancement of apoptosis, apoptotic nuclei were labelled using the nick end-labelling method. Numbers of labelled cells were markedly increased in the villus epithelium at 7–10 days p.i., while the numbers returned to normal 14 days p.i. when worms were rejected from the intestine and villus length became normal. Examination of the expression of the adhesion molecule E-cadherin showed granular immunoreactivity in the cytoplasm of atrophic villus epithelium with loss of normal localization to epithelial cell borders. In mast cell-deficient Ws/Ws rats, villus length was reduced as significantly as in +/+ counterparts at day 10 p.i. with marked increases in the numbers of apoptotic cells. These results suggested that villus atrophy was closely associated with enhanced apoptosis and loss of adhesion in epithelial cells. Mast cell activation appears not to be involved in these alterations.

The growth and morphology of the excretory system of Stephanurus dentatus were investigated using both the light microscope and the electron microscope. In the 3rd-stage larva a branched tubular system extends from the posterior end of the excretory ampulla. The subventral glands are two elongated cells in the body cavity, and these do not appear to be attached to the tubular system.

The tubular and glandular portions of the excretory system are intimately associated with each other at the posterior end of the ampulla or sinus from the 4th-stage, but throughout their development they remain histologically distinctive. Moreover, the nuclei of each portion of the system are of two different types; those of the tubular system are similar to hypodermal nuclei, whereas the nuclei of the subventral glands are like the intestinal nuclei.

The lateral canals are extensions of the excretory sinus and lie embedded in the hypodermal tissue of the lateral chords. As the worm matures, the lateral canals increase in relative length, and the anterior branch of each lateral canal develops during the 4th stage.

Pink viscous fluid which passes from the excretory pore is identical in appearance with the fluid which is found in the lateral chords. It is suggested that the fluid in the lateral canals gains access to the excretory system by way of canaliculi in the wall of the lateral excretory canal.

I am most grateful to Professor J. F. A. Sprent for his interest and advice during this investigation, and to Mr J. J. Mines and Mr C. Boel for their able technical assistance. I gratefully acknowledge the financial support given for this study by the Australian Dairy Produce Board and the U.S. Department of Health, Education and Welfare (No. A 107023–02).

Aspects of the ovicidal activity of the fungus Verticillium chlamydosporium Goddard on the eggs of Ascaris lumbricoides were observed in the laboratory using a scanning electron microscope. The hyphae of the fungus form structures which are considered to be penetration organs. The hyphae also form a network over the surface of the eggs. After penetration of the eggs, the hypha or hyphae ramify and the embryos are destroyed.

Experiments were designed to study the mating behaviour between the Schistosoma haematobium [male ]×S. intercalatum [female] hybrid and the 2 parental species S. haematobium and S. intercalatum. Individual worms were identified by electrophoretic analysis of glucose-6-phosphate dehydrogenase, which was characteristic for each isolate. Analysis of the data obtained showed that both heterospecific and homospecific pairs formed between the hybrids and S. haematobium and S. intercalatum. S. haematobium and the hybrid are better than S. intercalatum in forming pairs, and S. haematobium showed a greater homospecific mate preference compared with the hybrid. Analysis of the data using the Mantel-Haenszel test suggests that mating competition does exist between the schistosomes, with the hybrid being dominant over both the parental species and S. haematobium being dominant over S. intercalatum. The hybrid males showed a greater ability than S. intercalatum and S. haematobium males in taking away S. haematobium and S. intercalatum females from their homospecific males when introduced into a pre-established S. haematobium or S. intercalatum infection. They were able to take females from S. intercalatum homospecific pairs more easily compared with females from S. haematobium homospecific pairs. The significance of the results is discussed in relation to the epidemiological changes of schistosomiasis in Cameroon, where hybridization between S. haematobium and S. intercalatum has taken place, with S. haematobium and the hybrid managing to replace the endemic S. intercalatum over the last 30 years.

The bacterial flora of the intestine plays an important role in the virulence caused by Entamoeba histolytica. Cysteine proteinase (CP), an amoebic virulence factor, plays a major role in host cell destruction. The mechanism of increased virulence following bacterial co-association is not understood. We studied CP of E. histolytica HM1:IMSS which was co-associated with Escherichia coli K12 strain pre-incubated with GalNAc or CP specific inhibitor E 64. Co-association of E. histolytica with bacteria enhanced CP activity 3·6-fold as assessed by azocasein assay and substrate gel electrophoresis showed bands at molecular weights of 28, 35 and 56 kDa. Northern and Western blot analysis showed increase in ehcp2 and ehcp5 gene expression. Trophozoites co-associated with E. coli showed greater cytotoxicity of BHK cells by a 51Cr release assay than trophozoites that had not been co-associated; this enhancement was abolished by E-64 treatment. The killing of BHK 21 targets by E. histolytica was characterized by DNA laddering which was not inhibited with E-64. GalNAc pre-incubation of trophozoites reduced cytotoxicity and DNA laddering, while E. coli co-associated E. histolytica showed smearing with faint laddering of BHK implicating both necrosis and apoptosis. Hence, bacterial co-association increases CP activity and CP gene expression and contributes to the necrosis of the target cell.

The use of chemotherapy on a mass scale in endemic areas may lead to the appearance of resistant isolates through the mechanism of selective drug pressure. Studies have demonstrated that praziquantel (PZQ) is able to inhibit the excretory activity and to cause tegumental damage in Schistosoma mansoni adult worms. The use of the probe resorufin to evaluate excretory activity, as well as the probe Hoechst 33258 to detect tegumental damage in adult worms, may represent a method to identify resistant (or less susceptible) isolates. The purpose of the present work was to compare the changes caused by PZQ in the function of the excretory system and in the integrity of the tegument of adult worms from the LE isolate (susceptible to PZQ) and the LE-PZQ isolate (less susceptible to PZQ). Worms from the isolate LE-PZQ showed less severe tegumental lesions, in both in vitro and in vivo experiments, detected by labelling with Hoechst 33258 and continued to have a functional excretory system as shown by labelling with resorufin in vitro.

The balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malaria combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their β-O-glucosidic bond, become potent pro-oxidants. In this work we show that the glucosides inhibit the growth of Plasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of β-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD- erythrocytes. These results suggest that G6PD- infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD- erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD- bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD- genotype.

When adult Nippostrongylus brasiliensis were maintained in vitro they became damaged. Using the criteria of ultrastructural morphology, acetylcholinesterase isoenzyme pattern and the behaviour of the worms after transfer to a normal rat, this damage appeared to be similar to that produced by the in vivo action of antibodies.

Antibodies were shown to be responsible for the anterior migration of adult worms which occurs during primary infections in mature rats and in the prolonged infections seen in lactating and immature rats.

Antibody damaged worms and worms unaffected by antibodies were equally able to stimulate the immune response required for worm expulsion. Apparently antibody damage is not required for the initiation of the second immune component necessary for expulsion of this parasite.

Bile salts have been identified as an important trigger for excystation of Cryptosporidium oocysts but the hierarchy or synergism of this signal in relation to other triggers involved in excystation is poorly understood. In addition to excystation, bile salts have also been reported to increase the invasiveness of sporozoites within in vitro culture, possibly by affecting the secretory pathway via modification of intracellular calcium signalling. Nevertheless, incorporation of bile or bile salts into in vitro assays is not universal, with recent reports of negative effects on parasite growth. Here we report that bile and sodium taurocholate significantly affect both excystation rate and parasite in vitro growth. We demonstrate that their effect on excystation is dose, time and pre-treatment temperature dependent, while increases in parasite replication appear to be associated with modulation of parasite intracellular calcium and increased host cell susceptibility to infection. Notably, we illustrate that bile has a significant effect on host cells and can be cytotoxic at concentrations not much higher than those currently used for in vitro assays. This work should assist with more rational design of in vitro culture systems, with significant considerations for assay format when incorporating bile or bile salts as an excystation trigger.

Poekilostrongylus puertoricensis gen.nov., sp.nov. is proposed for nematodes recovered from Eleutherodactylus coqui, in Puerto Rico. The new genus is similar to Oswaldocruzia Travassos, 1917, but lacks longitudinal ridges on the cuticle. Oswaldocruzia lenteixierai Viqueras, 1938, is partially redescribed, and a key to the genera of the Oswaldocruziinae is given. Thelandros (Parathelandros) garciai sp.nov. is described from E. antillensis and E. portoricensis. Strongyloides sp. was found in E. antillensis and Aplectana spp. (females) were recovered from E. locustus, E. richmondi and E. coqui.

Camallanus marinus sp.nov. is described from eleven specimens recovered from a variety of marine fishes. It is the only species in Camallanus s.s. with a single spicule. Spinitectus palawanensis sp.nov. is described from two males and one female found in a scombrid fish. It is characterized by having 12–17 rings of 60–65 spines, a spicule ratio of 5:1, and ten pairs of caudal papillae. Cucullanus lutjani, sp.nov. is described from two specimens from lutjanid fishes. It is distinct in lacking a preanal sucker, in possessing a gubernaculum and eight pairs of cloacal papillae.Oceanicucullanus padfica gen. et sp.nov. is described from two specimens found in cyprinid fish. It is most similar to Indocucullanus, differing from it in the absence of a gubernaculum, and in possessing three pairs of teeth in the radii of the oesophagus. Philometra (Philometra) lateolabracis Yamaguti, 1935; Terranova sp.; and Procamallanus spp. are reported for the first time in the Philippines.

We wish to thank Dr D. S. Rabor, Silliman University, Damageute, Negros Oriental, Republic of the Philippines, for general support in field activities, and Dr Loren Wood, Curator of Fishes, and Dr Robert F. Inger, Curator of Reptiles, Chicago Museum of Natural History, for identification of hosts. Acknowledgement is also made to Dr Betty June Myers for technical assistance and advice in handling materials.

The plerocercoid larvae of the pseudophyllidean cestode Ligula intestinalie have been removed from the body-cavity of the roach, Rutilua rutilus, and cultured in vitro under aseptic conditions, using a technique similar to that successfully applied to Schistocephalus solidus.

In the larval condition, the genitalia are in a primitive, immature condition, with the testes, ovaries and yolk-glands only partially developed.

Larvae were cultured in peptone-broth in plugged tubes at a temperature of 40° C, i.e. the body temperature of birds in which the plerocercoids normally develop into adult worms.

Three of the nine larvae used became sexually mature after 7 days' cultivation, and oviposition took place. Copulation between successive genitalia of the same worm or with genitalia of another worm in the same culture tube was not observed.

Histological examination revealed that spermatogenesis, oogenesis, vitellogenesis and shell formation had apparently taken place in a normal manner. The testes contained giant polyploid cells as well as mature spermatozoa. The receptaculum seminis contained no spermatozoa, from which it was concluded that fertilization had not taken place. This was confirmed by the fact that attempts to hatch out the eggs were unsuccessful. The unfertilized eggs, however, underwent parthenogenic development within the uterus as far as the two-cell stage. Of the remaining six larvae, four developed sufficiently to undergo eversion of the cirrus, but died without oviposition taking place.

1. The distribution of glycogen and fat in Thelastoma bulhõesi has been shown to be essentially the same as in Ascaris lumbricoides.

2. The chief storage areas of glycogen are the muscle cells and the lateral lines, with smaller amounts in the dorsal and ventral lines, the intestine and the oesophagus.

3. The chief storage areas of fat are the dorsal, ventral and lateral lines, the intestine, the oocytes, oogonia and ova.

4. Fat has been found in the lumen of the intestine of several nematodes, especially in the males of Hammerschmidtiella diesingi after 24 hr. starvation.

5. Both glycogen and fat in females of T. bulhõesi were considerably reduced, except in the gonads, after 12 days starvation.

6. Staining whole mounts with oil red O was found to be the most convenient method of determining if the food reserves were being used or not.