Epidemiological data are often fragmented, partial, and/or ambiguous and unable to yield the desired level of understanding of infectious disease dynamics to adequately inform control measures. Here, we show how the information contained in widely available serology data can be enhanced by integration with less common type-specific data, to improve the understanding of the transmission dynamics of complex multi-species pathogens and host communities. Using brucellosis in northern Tanzania as a case study, we developed a latent process model based on serology data obtained from the field, to reconstruct Brucella transmission dynamics. We were able to identify sheep and goats as a more likely source of human and animal infection than cattle; however, the highly cross-reactive nature of Brucella spp. meant that it was not possible to determine which Brucella species (B. abortus or B. melitensis) is responsible for human infection. We extended our model to integrate simulated serology and typing data, and show that although serology alone can identify the host source of human infection under certain restrictive conditions, the integration of even small amounts (5%) of typing data can improve understanding of complex epidemiological dynamics. We show that data integration will often be essential when more than one pathogen is present and when the distinction between exposed and infectious individuals is not clear from serology data. With increasing epidemiological complexity, serology data become less informative. However, we show how this weakness can be mitigated by integrating such data with typing data, thereby enhancing the inference from these data and improving understanding of the underlying dynamics.

Allozyme markers were used to study genetic variation in Lecithochirium fusiforme within a natural population of Conger conger. Six of 16 enzyme-coding loci studied were found to be polymorphic. These loci were surveyed in 12 infrapopulations of adult flukes. High levels of genetic variation were detected (P=0·375); Ho=0·048; He=0·085). However, the population did not conform to Hardy-Weinberg expectations, as it showed a significant deficit of heterozygotes. L. fusiforme also exhibited low differentiation between infrapopulations (FST=0·064). Despite significant linkage disequilibrium at Pgm-1 and Pgm-2 (P<0·05), mating system does not appear to be the principal reason for the deficit of heterozygotes detected, because some polymorphic loci were in Hardy-Weinberg equilibrium. Association between FIS and FST statistics suggests the existence of the Wahlund effect. However, all infrapopulations showed a strong deficit of heterozygotes for most polymorphic loci (FIS=0·409). Detection of significant genetic differentiation among temporal samples and the existence of paratenic hosts in the life-cycle suggests the Wahlund effect, caused by the mixture of genetically distinct temporal samples in the infrapopulations. Occasional temporal gene flow also might explain the high estimated genetic polymorphism.

This personal review focuses on ways to approach and overcome some of the more common issues encountered while studying cestode zoonoses. The information presented here is based on the author's own experiences with immunological and molecular approaches for the detection of these parasites. There are many incongruities between immunological and molecular studies due to biased work. Nothing is perfect. Indirect approaches using either immunological, or even molecular tools, are limited without confirmation from direct evidence of infection. The dilemma of whether developing countries should develop their own diagnostic tests or rely on commercially available kits is also discussed.

Although co-infection is the norm in most human and animal populations, clinicians currently have no practical tool to assist them in choosing the best treatment strategy for such patients. Given the vast range of potential pathogens which may co-infect the host, obtaining such a practical tool may seem an intractable problem. In ecology the joint concepts of functional groups and guilds have been used to conceptually simplify complex ecosystems, in order to understand how their component parts interact and may be manipulated. Here we propose a mechanism by which to apply these concepts to pathogen co-infection systems. Further, we describe how these groups could be incorporated into a mathematical modelling framework which, after validation, could be used as a clinical tool to predict the outcome of any particular combination of pathogens co-infecting a host.

In the field of molecular and epidemiological parasitology, characterization of fast evolving genetic markers appears as an important challenge to consider the diversity and genetic structure of parasites. The study of respective populations can help us to understand their adaptive strategies to survive and perpetuate the species within different host populations, all trying to resist infection. In the past, the relative monomorphic features of Echinococcus multilocularis, the causative agent of alveolar echinococcosis and a severe human parasitic disease, did not stimulate studies dealing with the genetic variability of Echinococcus species or respective populations. A recently developed, characterized and validated original multilocus microsatellite, named EmsB, tandemly repeated in the genome, offered an additional opportunity for this line of investigation. We have compiled in this review new insights brought by this molecular tracker on the transmission activity of Echinococcus among different hosts and at different geographical scales.

Natural abundances of the stable isotopes, 15N/14N (δ15N) and 13C/12C (δ13C), were used to study temporal host-parasite relationships of the wild rabbit, Oryctolagus cuniculus (L.). During the 12-month sampling period, temporal isotopic shifts in δ15N were noted for dietary vegetation, host rabbit faeces and fur, but not for muscle or stomach contents. δ15N varied temporally for the parasitic cestode species, Mosgovoyia pectinata but not for Cittotaenia denticulata. Similarly, intestinal parasitic nematodes had apparent species-specific δ15N patterns. Only rabbit fur and intestinal parasitic nematodes did not exhibit temporal shifts in δ13C. Overall, host faeces and stomach contents were isotopically indistinct as a likely consequence of coprophagy. Relative to their host, parasitic nematodes were 15N-enriched, consistent with an increase in trophic level status. Conversely, cestodes were 15N-depleted. Isotopically, each parasite reflected a species-specific relationship with their rabbit host. This technique could be utilized to integrate parasites into food-web studies.

Oropsylla hirsuta is the primary flea of the black-tailed prairie dog and is a vector of the plague bacterium, Yersinia pestis. We examined the population genetic structure of O. hirsuta fleas collected from 11 prairie dog colonies, 7 of which had experienced a plague-associated die-off in 1994. In a sample of 332 O. hirsuta collected from 226 host individuals, we detected 24 unique haplotype sequences in a 480 nucleotide segment of the cytochrome oxidase II gene. We found significant overall population structure but we did not detect a signal of isolation by distance, suggesting that O. hirsuta may be able to disperse relatively quickly at the scale of this study. All 7 colonies that were recently decimated by plague showed signs of recent population expansion, whereas 3 of the 4 plague-negative colonies showed haplotype patterns consistent with stable populations. These results suggest that O. hirsuta populations are affected by plague-induced prairie dog die-offs and that flea dispersal among prairie dog colonies may not be dependent exclusively on dispersal of prairie dogs. Re-colonization following plague events from plague-free refugia may allow for rapid flea population expansion following plague epizootics.

We describe a new freshwater myxosporean species Ceratomyxa gracillima n. sp. from the gall bladder of the Amazonian catfish Brachyplatystoma rousseauxii; the first myxozoan recorded in this host. The new Ceratomyxa was described on the basis of its host, myxospore morphometry, ssrDNA and internal transcribed spacer region (ITS-1) sequences. Infected fish were sampled from geographically distant localities: the Tapajós River, Pará State, the Amazon River, Amapá State and the Solimões River, Amazonas State. Immature and mature plasmodia were slender, tapered at both ends, and exhibited vermiform motility. The ribosomal sequences from parasite isolates from the three localities were identical, and distinct from all other Ceratomyxa sequences. No population-level genetic variation was observed, even in the typically more variable ITS-1 region. This absence of genetic variation in widely separated parasite samples suggests high gene flow as a result of panmixia in the parasite populations. Maximum likelihood and maximum parsimony analyses placed C. gracillima n. sp. sister to Ceratomyxa vermiformis in a subclade together with Ceratomyxa brasiliensis and Ceratomyxa amazonensis, all of which have Amazonian hosts. This subclade, together with other Ceratomyxa from freshwater hosts, formed an apparently early diverging lineage. The Amazonian freshwater Ceratomyxa species may represent a radiation that originated during marine incursions into the Amazon basin that introduced an ancestral lineage in the late Oligocene or early Miocene.

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 (‘Human’) from 2 (‘Cattle’) in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

In order to study the infection dynamics of 2 renal myxozoans, Zschokkella hildae Auerbach, 1910 and Gadimyxa atlanticaKøie, Karlsbakk and Nylund, 2007 in cultured Atlantic cod, Gadus morhua L. aged 3–19 months, a specific single-round PCR assay and a double-label in situ hybridization protocol were developed. The results demonstrated that the 2 myxozoans show spatial separation of their development with regard to spore formation inside the renal tubules versus the collecting ducts and ureters, as well as temporal separation with Z. hildae proliferating and developing spores only once the G. atlantica infection decreases, despite the presence of both myxozoans in the smallest fry studied. These results strongly suggest within-host competition of the 2 myxozoans with potential suppression of Z. hildae by G. atlantica until G. morhua acquires immunity against G. atlantica. The quantification of the G. atlantica infection inside the renal tubules before and after a 29-day experimental growth performance study using fry from hatcheries with differing filtration systems showed that the intensity of infection with G. atlantica seems to be controlled if prolonged exposure to the myxozoan transmission stages takes place from hatching onwards. Surprisingly, growth rates in the trial were inversely affected suggesting that G. atlantica does not negatively influence cod fry growth performance.

A study has been carried out to determine the activity of genistein against adult liver fluke, Fasciola hepatica. Flukes were incubated in vitro in genistein at a concentration of 0·27 mg/ml (=1 mm). They ceased to move after 3 h, at which point the experiment was terminated and the specimens prepared for examination by scanning and transmission electron microscopy. Surface changes to the flukes comprised swelling and blebbing, especially in the posterior region of the flukes, and there was particular disruption to the spines, accompanied by some spine loss. Fine structural changes to the tegumental syncytium indicated an accelerated release of secretory bodies at the surface, but a reduction in their production within the cell bodies. Autophagic activity was evident in the tegumental cells, a phenomenon that was also observed in the gastrodermal cells. Disruption to the testis and vitelline follicles was severe, with an apparent block in the normal developmental sequence of the spermatogenic and vitelline cells, respectively. Shell protein production by the vitelline cells was also disrupted. In separate experiments, somatic muscle strips were exposed to concentrations of genistein ranging from 1 μm to 1 mm. There were statistically significant increases in the frequency and/or amplitude of muscle contractions at concentrations of 10 μm, 100 μm and 1 mm. The results suggest that genistein is capable of causing severe morphological and neuromuscular disruption to adult flukes in vitro over a short time-span.

Cyathostomins comprise a group of 50 species of parasitic nematodes that infect equids. Ribosomal DNA sequences, in particular the intergenic spacer (IGS) region, have been utilized via several methodologies to identify pre-parasitic stages of the commonest species that affect horses. These methods rely on the availability of accurate sequence information for each species, as well as detailed knowledge of the levels of intra- and inter-specific variation. Here, the IGS DNA region was amplified and sequenced from 10 cyathostomin species for which sequence was not previously available. Also, additional IGS DNA sequences were generated from individual worms of 8 species already studied. Comparative analysis of these sequences revealed a greater range of intra-specific variation than previously reported (up to 23%); whilst the level of inter-specific variation (3–62%) was similar to that identified in earlier studies. The reverse line blot (RLB) method has been used to exploit the cyathostomin IGS DNA region for species identification. Here, we report validation of novel and existing DNA probes for identification of cyathostomins using this method and highlight their application in differentiating life-cycle stages such as third-stage larvae that cannot be identified to species by morphological means†.

Interferon-gamma (IFN-γ) has a crucial role for host defence against parasite infection. It is not clear, however, how IFN-γ affects the parasite-infected host cells. The effect of IFN-γ on Neospora caninum-infected cells was investigated in murine fibroblasts and canine kidney cells in vitro. In the presence of IFN-γ, the viability of the infected host cell was decreased and apoptotic cell death occurred, as analysed by DNA stainings with propidium iodide and a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) and DNA fragmentation. The percentage of apoptotic cells depended on the dose of IFN-γ. Flow cytometric analysis indicated a significant increase of FasL expression on the IFN-γ-treated cells following N. caninum infection. Moreover, IFN-γ treatment down-regulated Bcl-2 expression in the cells cultured with N. caninum while parasite infection up-regulated Bcl-2 expression. The present study suggests that the IFN-γ induced increases of FasL expression and down-regulated Bcl-2 expression in N. caninum-infected cells are associated with apoptosis in vitro.

Plasmodium falciparum, the agent responsible for malaria, is an obligate intracellular protozoan parasite. For proliferation, differentiation and survival, it relies on its own protein-encoding genes, as well as its host cells for nutrient sources. Nutrients and subsequent metabolites are required by the parasites to support their high rate of growth and replication, particularly in the intra-erythrocytic stages of the parasite that are responsible for the clinical symptoms of the disease. Advances in mass spectrometry have improved the analysis of endogenous metabolites and enabled a global approach to identify the parasite's metabolites by the so-called metabolomic analyses. This level of analysis complements the genomic, transcriptomic and proteomic data already available and should allow the identification of novel metabolites, original pathways and networks of regulatory interactions within the parasite, and between the parasite and its hosts. The field of metabolomics is just in its infancy in P. falciparum, hence in this review, we concentrate on the available methodologies and their potential applications for deciphering important biochemical processes of the parasite, such as the astonishingly diverse phospholipid biosynthesis pathways. Elucidating the regulation of the biosynthesis of these crucial metabolites could help design of future anti-malarial drugs.

The entire ribosomal DNA repeat unit of a steinernematid species (Nashes isolate) was cloned as three separate EcoR I fragments in the plasmid pUC18. An equimolar cocktail of these three clones was used to identify Steinernema species on Southern blots as each species displays its own unique restriction fragment length polymorphisms. The clones also identified two new species isolated in a soil survey of coastal regions of Britain. One of the clones (pSn4.0) can detect length heterogeneities in the rDNA repeat unit of various isolates of some of the species, particularly the most common in the United Kingdom, S. feltiae. These differences in the rDNA repeat unit length remained constant over several years for one isolate of S. feltiae, but were different for each of the geographical isolates studied to date.

The nature of antigens present on the surface coat of Trypanosoma brucei brucei (serotype AnTat–1) trypanosomes has been investigated by light and electron microscopy using indirect immunofluorescence and immunoenzymatic labelling techniques. The use of a monospecific anticomponent V serum has shown that the variant-specific antigen, component V, is the major constituent of the surface coat. Antigens of heterologous serotype, of culture form trypanosomes, of host serum proteins or host red blood cells were not demonstrated on the surface coat.

1. Measurements were made of the head length, head width, total body length, antenna length, length and breadth of 3rd antennal segment of head and body lice from natural infestations. In all measurements (except the last) the means were significantly different for. the two forms, but in all cases there was considerable overlap.

2. Head lice reared continuously on the body for forty-three generations (over 2 years) did not change systematically in size. Body lice reared under similar conditions were likewise constant.

3. There was a highly significant difference between mean measurements of all the samples of head lice reared for up to 2 years on the body when compared with body lice reared for a similar period in the same way. The means for lice worn 24 hr. per day were less than those worn 12 hr. per day in both strains. An experiment showed that this is probably due to a difference in average temperature.

4. The mean live weights of head and body strains were even more distinct than linear dimensions on the several occasions when these were recorded.

5. Measurements of the dimensions of the opercula of the eggs of the two races and the numbers of air cells in them were distributed in the same way as the body measurements: that is, distinct means but overlapping individuals.

6. Ventral abdominal muscles in wild head lice are typically confined to segment 5. (The forty specimens examined adhered to this rule.) In wild body lice, muscles are usually also present in segment 4 as well.

7. Hybrids (female body x male head louse) were fertile for several generations. In size, the F1 generation was intermediate, but the F2 and F3 generations became identical with the body louse strain.

8. Eggs of the body louse strain hatched at the same time as those of the head strain, but the body strain reached a significantly higher percentage hatch.

9. The times of moulting during development were studied in both races in relation to differing opportunities to feed (24, 12, 6 and 3 hr. per day). The two strains showed very similar reactions in regard to speed of development, but throughout there was a higher pre-imaginal mortality in the head strain.

10. Head lice were shown to be considerably more susceptible to starvation than body lice.

11. If adults of the two strains are mixed, there is no tendency towards homogeneous mating. Of the mixed matings, those between body louse males and head louse females are much more common than the reverse.

12. Female head lice show a slight difference in their egg-laying preferences when offered a choice of hair, voile and tape.

My thanks are due to Prof. P. A. Buxton, F.R.S., for a number of helpful suggestions based on his extensive knowledge of louse biology and to Dr J. O. Irwin for critical comments on the statistical issues.

The physiology of the neuromuscular systems in Hymenolepis diminuta and H. microstoma was studied in vitro using intact, adult worm and strips of worm body wall. Intact worms were insensitive to ionic changes in the in vitro buffering system. However, strips of body wall containing longitudinal muscles were extremely sensitive to ionic manipulation. In intact worms tension generated in the strobila had two components; small brief tension peaks up to 500 mg amplitude are superimposed on larger, longer peaks of up to 1200 mg amplitude. Removal of the scolex and neck region either failed to show significant changes in tension, or showed a reduction in amplitude but not of frequency. Muscle contraction of both H. diminuta and H. microsoma were qualitatively similar. In split-worm preparations the concentration of Ca2+ in the bathing solution significantly affected both spontaneous and evoked contractions in H. diminuta and H. microstoma; the addition of CaCl2 greatly reduced the amplitude and frequency of the contractions. The chloride salts of cobalt, barium, cadmium and manganese elicited prolonged contractions of the longitudinal musculature of both H. diminuta and H. microstoma. While CoCl2 was the most effective in stimulating muscle contraction, the magnitude of the response varied with the concentration of Ca2+ in the bath. The results indicate that peripheral inhibition is extremely important in cestode motor control and that extracellular calcium ions may regulate the peripheral inhibitory mechanisms.

The biology and life-cycle of Holostephanus lühei is briefly described. The similarities in morphology between the adhesive organ gland cells of H. lühei and Cyathocotyle bushiensis are stressed. Alkaline and acid phosphatases were demonstrated in the adhesive organ gland cells and cuticle. Acid phosphatase also occurs in the caecal cells. Non-specific esterase, sensitive to E600 10−5M and Mipafox 10−3M, is present in the gland cells. The caeca contain a non-specific esterase sensitive to AgNO3 10−2M and PCMB 10−4M. Leucine aminopeptidase is present in the gland cells. The in vitro studies confirmed that the secretory products pass to the exterior of the parasite and have a histolytic action.

The four strigeids studied, Cyathocotyle bushiensis, Holostephanus lühei, Diplostomum spathaceum and Apatemon gracilis minor, are compared and the biological role of the secreted enzymes are discussed.

My sincere thanks are due to Dr D. A. Erasmus for suggesting this line of investigation and for his encouragement during the study. I am also grateful for his performing, on my behalf, the infection experiments and criticizing the manuscript. I wish to thank Professor J. Brough for his interest in the progress of the work, and the University College, Cardiff, for a grant from William E. Morgan's Bequest.

(i) Amoebae closely resembling Entamoeba coli (of Man) have been studied in macaques (M. rhesus, M. sinicus, M. nemestrinus) and in a marmoset (Hapale jacchus).

(ii) Pure strains of these amoebae have been isolated and cultivated, and carefully compared with similar pure strains of E. coli from Man (E. coli hominis).

(iii) The complete life-cycles of these various strains—amoebae, cysts, and all stages of precystic and metacystic development—have been studied in vitro.

(iv) The “E. coli” of macaques (E. c. macacorum) was experimentally transmitted to a man, who ingested pure culture-cysts of a strain derived from M. rhesus.

(v) The “E. coli” of the marmoset (E. c. jacchi) was likewise transferred to M. sinicus, and from this host again to M. rhesus and Man.

(vi) The natural infections observed in M. sinicus, M. rhesus, and H. jacchus all died out spontaneously after persisting for various periods (4½ years, 2 years, and 1 year respectively). The experimental infection with E. coli macacorum induced in Man also disappeared spontaneously (after about 6 weeks).

(vii) Attempts to infect kittens experimentally with E. coli jacchi were uniformly unsuccessful.

(viii) All infections with “E. coli” from every source—whether natural or experimental—have proved to be completely innocuous.

(ix) Culture-cysts of various strains of “E. coli” have been found able to live at low temperatures for all times up to about 4½ months (maximum 135 days at 1°–2° C., for a strain from H. jacchus).

(x) The “E. coli” of H. jacchus—and all its experimental derivatives—ingests human red blood-corpuscles in vitro as readily as E. histolytica.

(xi) Several strains of E. coli hominis, isolated directly from naturally infected human beings, have been found to possess the same property.

(xii) Since strains of E. coli thus exist—both in men and in monkeys—which eat human red corpuscles avidly, this faculty can no longer be considered as peculiarly characteristic of E. histolytica; and the importance of this observation, for the diagnosis of amoebic dysentery, is therefore emphasized. The statement that E. histolytica ingests red corpuscles, while E. coli does not, is untrue. It is not possible to distinguish these species by this character alone.

(xiii) As it has been found impossible to distinguish various strains of “E. coli” living in macaques, a marmoset, and men, by any recognizable specific feature—all of them being so closely alike morphologically, physiologically, culturally, and in their ability to inhabit these different hosts—it is concluded that no specific difference actually exists between any of them. The “E. coli” of the Macaques and of the Common Marmoset is probably Entamoeba coli itself—the species which naturally inhabits Man.