Captive-bred Mus musculus (house mice) and Apodemus sylvaticus (field mice) were each infected with 50 oocysts of Toxoplasma gondii M1 strain per os and infection in them and their offspring was assessed by polymerase chain reaction (PCR) amplification of the T. gondii B1 gene in brain tissue and by serology, using the modified agglutination test (MAT). The chronically infected female A. sylvaticus (n=10) and M. musculus (n=23) were mated at least 6 weeks after infection (and subsequently to produce up to 6 litters) and their pups examined 3 weeks after weaning at 6 weeks of age. By PCR, in offspring of A. sylvaticus and M. musculus respectively, vertical transmission was demonstrated in 82·7% (n=83) and 85·0% (n=207) of all pups (N.S., P>0·05), 95% (n=21) and 100% (n=30) of all litters (N.S., P>0·05), with a mean (±S.E.) proportion of each litter infected of 0·87 (0·06) and 0·86 (0·04) (N.S., P>0·05). There was no change in any of these variables between first and subsequent litters. By serology, whilst MAT suggested 100% vertical transmission in A. sylvaticus, it under-estimated rates of infection in offspring of M. musculus. A limited series of bioassays from M. musculus tissues confirmed the good correlation of PCR and the poor correlation of MAT with mouse inoculation. These results indicate that vertical transmission in A. sylvaticus and M. musculus is extremely efficient and probably endures for the life of the breeding female. This mechanism favours parasite transmission and dispersion by providing a potential reservoir of infection in hosts predated by the cat.

Promastigotes of Leishmania braziliensis, L. donovani, L. major and L. mexicana recently derived from tissue amastigotes were cultured in Schneider's Drosophila medium supplemented with 20% (v/v) heat-inactivated foetal calf serum and 25 μg gentamicin sulfate/ml at pH 5·5. These cultures produced more metacyclic promastigotes in their stationary-phase populations than others cultured at pH 7·0. Metacyclic promastigotes possessed a short ([les ]8 μm) and narrow ([les ]1·5 μm) cell body with a flagellum twice or more the length of the cell body. Promastigotes from acidic cultures were more resistant to complement-mediated lysis and more infective in vivo than those grown at neutral pH. These results demonstrate that induction of metacyclogenesis by acidic pH is a response conserved across a variety of species of Leishmania.

The first internal transcribed spacer (ITS1) of the nuclear ribosomal DNA repeat was sequenced for members of the Schistosoma japonicum species complex (S. malayensis, S. mekongi and 2 geographical isolates of S. japonicum). The ITS1 is composed of 3 distinct regions: the 5′ end (23 nucleotides); a tract of approximately 90–140 nucleotides, which occurs up to 7 times in tandem, the number varying even within an individual in all species investigated in this study; the 3′ region (378 nucleotides), which lacks repeats. There is size and sequence variation among copies of the ITS1 repeat within a single individual. The relative abundances of size variants of ITS1 in S. japonicum have been ascertained by hybridizing genomic digests with an ITS1 probe. Multiple repeats and intra-individual variation in numbers and abundance of these is a feature of the Asian schistosomes, but not generally of African schistosomes. Possible reasons for this difference in ITS1 between African and Asian schistosomes are discussed. The ITS1 repeat sequences described for African schistosomes are different to, and cannot be aligned with, those from the Asian species described here, whereas the remainder of the ITS1 can be aligned quite easily.

The Zn2+-chelating metalloprotease inhibitor 1,10-phenanthroline (phenanthroline, 5–150 μM) elicited dose-dependent contraction of the longitudinal and circular (transverse) musculature of adult male schistosomes. At the same concentrations, phenanthroline did not cause contraction of dispersed individual muscle fibres. The phenanthroline-induced contractions were reduced by the inclusion of 100 or 300 μM Zn2+ in the extracellular medium. Phenanthroline (0·5–150 μM) also inhibited the egg production of adult worm pairs in vitro, with a 98% reduction at 50 μM. When worm pairs were exposed to phenanthroline, the males detached from the dish and released the females, resulting in unpaired worms. At the higher concentrations (50 and 150 μM), the worms were killed in vitro. Worm burdens were reduced by over 50% in infected mice injected with phenanthroline (20 mg/kg/day for 4 days), but twice the dose resulted in only a 25% reduction. Phenanthroline injections also induced an hepatic shift and an unpairing of adult worms in infected mice, and the female worms appeared degenerate and lacked gut pigmentation. Mice fed a diet containing 0·3% phenanthroline received significant protection from infection when challenged with schistosome cercaria, where phenanthroline-fed mice had 94% fewer adult worms than control mice. The broad range of phenanthroline effects on schistosomes suggests broad and important functions for metalloproteases in these worms.

In the savanna areas of tropical Africa, cattle are frequently infected with the filaria Onchocerca ochengi. This parasite is closely related to Onchocerca volvulus, the causative agent of human onchocerciasis (river blindness), and is capable of developing in the same vector, Simulium damnosum s.l. In North Cameroon, where both O. ochengi and O. volvulus are endemic, we carried out a field study (reported in this and 2 following papers) to examine to which extent the transmission of the 2 parasite species overlap and what influence this has on the epidemiology of human onchocerciasis. In this paper we report our experiments to determine which of the S. damnosum species in North Cameroon act as vectors of O. ochengi, how efficiently they do so and whether other Simulium species play a vector role. To this end, infected cattle were exposed near 5 rivers in different geographical areas. Among 14 Simulium species identified as aquatic and/or adult stages at these rivers, only 6 (S. squamosum, S. damnosum s.s., S. sirbanum, S. bovis, S. wellmanni and S. hargreavesi) were found to bite cattle in important numbers in at least 1 of the sites. The 3 species of the S. damnosum complex were all capable of ingesting microfilariae (mf) of O. ochengi and developing a proportion of them to infective larvae (L3). Whereas S. squamosum and S. damnosum s.s., the prevailing vectors in the Guinea and Sudan savanna respectively, showed a high vector competence (17% of ingested mf developed to L3), S. sirbanum, which was much rarer in both areas, appeared to have a much lower susceptibility (2%). Other boophilic Simulium species were only seen in certain sites and seasons, being either incapable of ingesting important numbers of O. ochengi mf from body regions where these mf were abundant (S. bovis, S. hargreavesi); not able to support the development of ingested mf to L3 (S. wellmanni), or bit cattle preferentially in the ears, where O. ochengi mf do not occur (S. hargreavesi). We conclude that in North Cameroon members of the S. damnosum complex are the only important vectors of O. ochengi, with S. squamosum and S. damnosum s.s. being the main vectors.

In order to assess the prevalence of the cattle filaria Onchocerca ochengi in onchocerciasis vectors (Simulium damnosum s.l.) in North Cameroon, we searched for a means to morphologically identify its developing larvae, which closely resemble those of O. volvulus. To this end microfilariae of the 2 Onchocerca species were isolated from slaughter cattle in Ngaoundéré and injected into neonate Simulium species. Whereas the early developmental stages (sausage stage, L2 and pre-infective larva) were indistinguishable, the infective larvae (L3) of O. ochengi were longer (median: 740 μm), more slender (diameter = 19·3 μm = 2·6% of body length) and had a relatively shorter tail (4·9% of body length) than those of O. volvulus (680 μm, 20·5 μm, 3·0% and 5·8% respectively). The tail of O. ochengi L3 was thick and rounded, whereas it was slightly tapering in O. volvulus L3. O. ochengi L3 produced by feeding flies on infected cattle in a different area in North Cameroon (Sora Mboum) showed the same features as intrathoracically produced O. ochengi L3 from Ngaoundéré, but were even longer (785 μm). On the basis of the differences in length, relative diameter, length of the tail and shape of the tail, a simple key for the separation of O. volvulus and O. ochengi L3 was elaborated, and 248 L3 found in wild S. damnosum s.l. were separated into ‘O. ochengi’ (160 L3) and ‘O. volvulus’ (88 L3) following this key. Sequential dot blot hybridization of each of the 248 larvae with a DNA probe which reacts with O. ochengi and O. volvulus but not with other Onchocerca species (pOo5/1) and with an O. volvulus-specific DNA probe (pOv12) revealed that the morphological identification had been correct in 86–91% of the cases. Only a small proportion (6–9%) of the dot blots did not react with either probe. Since this proportion was equal in experiments using experimentally produced L3 and in experiments using wild L3, the non-hybridization was certainly due to a loss of L3 during washing of the filters and not due to the presence of other unknown L3 species resembling O. volvulus and O. ochengi. Our study shows that in Cameroon it is possible to identify O. volvulus and O. ochengi infective larvae during routine fly dissections by morphology alone.

In North Cameroon, the vector of Onchocerca volvulus (causative agent of human onchocerciasis) also transmits 2 filariae of animals: O. ochengi from cattle and O. ramachandrini from wart hogs. In order to assess the qualitative and quantitative roles of these ‘animal filariae’ in the epidemiology of O. volvulus, the transmission of the 3 parasites was measured in 2 villages and related to the endemicity of human onchocerciasis. In Galim, a cattle-farming Guinea savanna village where wild animals are rare, the overwhelming majority of all filarial infections found in the Simulium damnosum s.l. vectors throughout the year were O. ochengi (89%). The remaining infections were mainly O. volvulus (10·5%), and a few O. ramachandrini (0·5%). In Karna, a crop-farming Sudan savanna village where cattle are rare, but wild animals common, flies were also more frequently infected with animal filariae than with the human parasite. In the dry season, when nomadic cattle are present, 54% of all infections were O. ochengi, 36% O. volvulus and 10% O. ramachindrini. In the rainy season, when the cattle move away, flies were mainly infected with O. ramachandrini (52% of all infections) and secondly with O. volvulus (48%). In Karna, the relationship between the Annual Transmission Potential (ATP) of O. volvulus and its prevalence in the human population conformed to other onchocerciasis foci, in that a moderate ATP led to hyperendemic onchocerciasis. In Galim, however, a 7-fold higher O. volvulus-ATP (caused by a very high biting rate of the flies) contrasted with a strikingly low endemicity of onchocerciasis. Since, at the same time, in Galim the transmission of O. ochengi (measured on man) was very high (15000 L3/fly collector/year), we hypothesize that the reduced endemicity of onchocerciasis in Galim is due to ‘natural heterologous vaccination’ by the large annual number of O. ochengi-L3, inoculated into man by anthropo-boophilic S. damnosum s.l. The importance of micro-epidemiology for the understanding of the interlinkage of human and animal onchocerciasis is discussed.

Echinococcus multilocularis is a cestode parasite that predominantly infects red and arctic foxes as definitive hosts. Ingestion of E. multilocularis eggs and subsequent post-oncospheral infection with the larval stage (metacestode) of the parasite results in alveolar echinococcosis (AE), a life-threatening hepatic disease concerning humans and other intermediate hosts such as small rodents. The primary fluid-filled vesicles of the asexually proliferating metacestode are comprised of an inner germinal layer, a syncytial tegument, and an outer, acellular, so-called laminated layer. This laminated layer may play an important role in protecting the developing E. multilocularis metacestode from host immune reactions, and laminated layer-associated components represent potential targets for intervention during the course of AE. We have used an in vitro cultivation technique for the long-term maintenance and proliferation of E. multilocularis metacestodes in order to generate premature (protoscolex-free) parasite vesicles. A polyclonal antiserum was raised against this host-free parasite tissue. Subsequent immunoblot analysis of parasite fractions obtained by Triton X-114 extraction led to the identification of a 116 kDa protein (named EmP2) within the Triton-insoluble fraction. The characterization of EmP2 by SDS–PAGE, Western blotting, and by immunofluorescence revealed that EmP2 is a laminated layer-associated protein.

The formation of the definitive procercoid excretory system of the pseudophyllidean cestode Triaenophorus nodulosus has been investigated. This process can be divided into 3 main stages. In the first stage, active autophagic processes lead to the formation of intracellular excretory canals. In the second stage, the process of the autolysis is enhanced and the system of intercellular lacunae functioning as an excretory system is formed. A definitive excretory system of the procercoid forms at the next stage, by means of the migration and proliferation of undifferentiated cells replacing the lacunar system.

Full-length cDNAs encoding cytosolic (SODc) and putative extracelluar (SODe) Cu/Zn superoxide dismutases (SODs) from the ovine gastrointestinal parasitic nematode Haemonchus contortus have been isolated and characterized. The predicted sequences of the H. contortus SODs showed strong homology to other helminth SODs, the highest level of sequence similarity was with those of the free-living nematode Caenorhabditis elegans. The predicted amino acid sequence of the putative extracellular form contained an N-terminal extension with the characteristics of a signal sequence including a potential signal peptidase cleavage site. Transcripts of both classes of Cu/Zn SOD were detected in all life-cycle stages examined. The cytosolic SOD mRNA was approximately 6-fold more abundant than that of the extracellular enzyme in adult parasites. Immunoblotting with antisera raised to in vitro-expressed parasite SODs revealed the presence of 2 proteins in extracts of adult H. contortus, with molecular masses of approximately 19·8 and 18 kDa. An additional protein of approximately 16·8 kDa was detected in adult ES material. Immunofluorescent staining showed Cu/Zn SOD was localized in the body wall musculature and the pharynx in adult worms and in the uterine tract of adult females. The immunogenic properties of recombinant H. contortus Cu/Zn SODs was assessed in a challenge infection experiment in lambs.

In most real-world contexts the sampling effort needed to attain an accurate estimate of total species richness is excessive. Therefore, methods to estimate total species richness from incomplete collections need to be developed and tested. Using real and computer-simulated parasite data sets, the performances of 9 species richness estimation methods were compared. For all data sets, each estimation method was used to calculate the projected species richness at increasing levels of sampling effort. The performance of each method was evaluated by calculating the bias and precision of its estimates against the known total species richness. Performance was evaluated with increasing sampling effort and across different model communities. For the real data sets, the Chao2 and first-order jackknife estimators performed best. For the simulated data sets, the first-order jackknife estimator performed best at low sampling effort but, with increasing sampling effort, the bootstrap estimator outperformed all other estimators. Estimator performance increased with increasing species richness, aggregation level of individuals among samples and overall population size. Overall, the Chao2 and the first-order jackknife estimation methods performed best and should be used to control for the confounding effects of sampling effort in studies of parasite species richness. Potential uses of and practical problems with species richness estimation methods are discussed.