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.

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.

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.

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.

Attenuated lines of Eimeria acervulina were isolated between 62 and 72 h post-infection from the Houghton (H) strain. The inoculation of small numbers of oocysts of precocious (HP) lines gave substantial protection to Light Sussex chicks kept on litter against challenge with the virulent H strain. The precocious trait of the 72 h HP line was shown to be stable because the kinetics of oocyst production remained unaltered after 9 consecutive passages through birds in which only late developing oocysts were used for passage. The precocious 62 h HP line was subjected to further selection for 5 consecutive passages. The resultant (62 A HP) line was shown to be more attenuated than the 72 h HP line but remained capable of immunizing chicks against challenge with the H strain.

Proteinases released during in vitro maintenance of third (L3) and fourth larval stage (L4) and adult Teladorsagia circumcincta (formerly Ostertagia circumcincta), an ovine abomasal nematode parasite, were characterized on the basis of pH optima, molecular size and specific proteinase inhibitor sensitivity. Enzyme activity was maximal at alkaline pH and stage-specific release was demonstrated. Proteinases released by the adult parasite degraded a variety of protein substrates including plasminogen, albumin and haemoglobin, in a pH-dependent manner. At alkaline pH fibrinogen degradation was restricted to the α and β peptide chains although the γ peptide chain was also degraded at acidic pH. Inhibitor sensitivity studies indicated that degradation was predominantly due to metalloproteinases although aspartyl proteinase activity was indicated at acidic pH.

The cercaria of Fasciola hepatica has four types of cystogenic cells which can be characterized by their position and by their histochemical properties. On the basis of their histochemical reactions, each type of cell was related to a specific layer in the cyst wall of the metacercaria.

The tanned protein cells occupy almost all of the ventral half of the body and contain large granules consisting of basic protein and phenols. Although the presence of a phenolase could not be demonstrated, it is considered that the granules are composed of tanned protein. Before the cercaria leaves the redia, the granules are secreted to the exterior to form a layer contained by a thin, bounding membrane. The tanned protein cells form layer I of the cyst wall.

The mucopolysaccharide cells are concentrated at the dorsal and dorso–lateral surface except for a few around the ventral sucker. They contain acid and neutral mucopolysaccharides and contribute to layers II and IIIb and c. Some of the granules are secreted to the exterior before the cercaria leaves the redia, in a similar manner to the tanned protein granules.

The mucoprotein cells are few in number and lie centrally between the ventral sucker and the pharynx. They give reactions for proteins and carbohydrate-protein complexes and probably help to form layers II and IIIa of the cyst wall.

The keratin cells fill almost the entire dorsal half of the body and contain numerous rod-like granules. These rods are composed of a protein containing sulphydryl groups and disulphide bonds and give rise to layer IV. After secretion of the tanned protein granules to the exterior, some of the keratin cells move to the ventral part of the body formerly occupied by the tanned protein cells.

The outer cyst wall exists in a preformed state in the free swimming cercaria.

I am grateful to Professor J. D. Smyth and Dr J. A. Clegg of this department for advice and encouragement during this investigation and in the preparation of the manuscript. The work was done during the tenure of a Commonwealth Post-Graduate Scholarship.

Distributions of the intensities of helminth infections within their host populations are invariably aggregated. In the case of the intestinal nematodes, the degrees of aggregation have been shown to be species specific, and constant for any given species despite geographical variation in study sites. This species-specific aggregation can be quantified and used as a tool in planning control interventions. One practical application is that the prevalence of infection can be used to predict the prevalence of heavy infection and thus the risks of morbidity. This paper investigates the patterns of aggregation in schistosome egg counts in different endemic areas in Africa (data sets were obtained from Burundi, Cameroon, Tanzania, Zambia and Zaire). The analysis demonstrates that the degree of parasite aggregation, for both Schistosoma mansoni and S. haematobium, differs amongst the different study localities. This is probably due to area-specific differences in host exposure and immunity. This implies that for these schistosome species, it is not possible to predict egg count distributions or morbidity levels from prevalence data alone.

A dipeptidylpeptidase (DPP) was isolated from Fasciola hepatica by gel-filtration and ion-exchange chromatography. The exoproteinase is secreted by newly excysted juveniles, immature and mature flukes. The liver fluke DPP is a serine proteinase of molecular weight > 200 kDa and differs from previously characterized mammalian DPPs in its substrate preference and susceptibility to inactivation by inhibitors. The parasite DPP may function in the latter stages of the proteolytic digestion of host macromolecules. In this manner, the enzyme may be important in providing the parasite with dipeptides that could be absorbed through the intestine as nutrient.

Trypanosomatids of the genera Angomonas and Strigomonas (subfamily Strigomonadinae) have long been known to contain intracellular beta-proteobacteria, which provide them with many important nutrients such as haem, essential amino acids and vitamins. Recently, Kentomonas sorsogonicus, a divergent member of Strigomonadinae, has been described. Herein, we characterize the genome of its endosymbiont, Candidatus Kinetoplastibacterium sorsogonicusi. This genome is completely syntenic with those of other known Ca. Kinetoplastibacterium spp., but more reduced in size (~742 kb, compared with 810–833 kb, respectively). Gene losses are not concentrated in any hot-spots but are instead distributed throughout the genome. The most conspicuous loss is that of the haem-synthesis pathway. For long, removing haemin from the culture medium has been a standard procedure in cultivating trypanosomatids isolated from insects; continued growth was considered as an evidence of endosymbiont presence. However, we demonstrate that, despite bearing the endosymbiont, K. sorsogonicus cannot grow in culture without haem. Thus, the traditional test cannot be taken as a reliable criterion for the absence or presence of endosymbionts in trypanosomatid flagellates. It remains unclear why the ability to synthesize such an essential compound was lost in Ca. K. sorsogonicusi, whereas all other known bacterial endosymbionts of trypanosomatids retain them.

Establishing a health screening protocol is fundamental for successful captive breeding and release of wildlife. The aim of this study was to undertake a parasitological survey focusing on the presence of trypanosomes in a cohort of Regent Honeyeaters, Anthochaera phrygia, syn. Xanthomyza phrygia (Aves: Passeriformes) that are part of the breeding and reintroduction programme carried out in Australia. We describe a new blood parasite, Trypanosoma thomasbancrofti sp. n. (Kinetoplastida: Trypanosomatidae) with prevalence of 24·4% (20/81) in a captive population in 2015. The sequence of the small subunit rRNA gene (SSU rDNA) and kinetoplast ultrastructure of T. thomasbancrofti sp. n. are the key differentiating characteristics from other Trypanosoma spp. T. thomasbancrofti sp. n. is distinct from Trypanosoma cf. avium found in sympatric Noisy Miners (Manorina melanocephala). The SSU rDNA comparison suggests an intercontinental distribution of T. thomasbancrofti sp. n. and Culex mosquitoes as a suspected vector. Currently, no information exists on the effect of T. thomasbancrofti sp. n. on its hosts; however, all trypanosome-positive birds remain clinically healthy. This information is useful in establishing baseline health data and screening protocols, particularly prior to release to the wild.

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.

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.

1. The development of Theileria parva in engorged larvae of R. appendiculatus exposed to a temperature of 4–6° C. for 3 days at intervals of 2, 4, 6, 8 and 9 days after repletion is not retarded; and it appears, from previous experiments and those now described, that, as long as the tick survives, climatic conditions do not kill or weaken the parasite.

2. The virulence of East Coast fever transmitted by ticks fed in the tail brush did not differ from the disease conveyed by ticks infesting the ears.

3. Reference is made to records of mild reactions to Bast Coast fever when the parasites are either rare or absent; and instances are given of such reactions followed by recovery in experimental animals. Although the mild form of the disease seems associated at times with light tick infestation, it is proved that a few infected ticks also transmit a fatal East Coast fever. It is shown also that ticks fed on a bovine during a short or mild reaction can produce a virulent form of the disease in susceptible animals, and that mild reactors acquire an immunity to the virulent disease.

4. The experiments which are described indicate that ticks do not become infected with T. parva from the blood of a bovine for the first 4 days of the reaction period.

5. Evidence is produced which strongly suggests that T. parva tends to disappear from infected hungry ticks kept under laboratory conditions for about a year or more. The age of the tick would appear to be an important factor in the transmission of East Coast fever.

6. Attempts to break down the immunity to East Coast fever, and to produce “turning-sickness”, by massive infestations of ticks were unsuccessful; and further experiments on the transmission of East Coast fever by ticks which had fed on animals suffering from “turning-sickness” did not confirm earlier positive results.

These conflicting results may be explained by the observations that “turning-sickness”, associated with the blocking of the small capillaries of the brain by lymphocytes containing schizonts indistinguishable from the Koch's bodies of T. parva, may be divided into three stages, namely, acute, subacute and chronic; and it is suggested that infection of ticks with T. parva from “turning-sickness” could normally be successful only from animals in the acute and early part of the subacute stages since piroplasms disappear from the peripheral blood stream in the more advanced cases of “turning-sickness”.

7. The opinions of other workers on East Coast fever are discussed in the light of the experiments carried out at Kabete and of field experience in Kenya Colony. It is explained how the results can be applied to the movement of cattle from endemic areas over country free from East Coast fever.

Alterations in the hepatic portal vasculature of NMRI mice infected with Schistosoma mansoni were assessed using a microsphere injection technique. The accumulation of eggs in the livers of infected mice and the development of portal hypertension were closely related to the worm pair burden during the first 15 weeks of infection. Individual variation between mice harbouring identical patent worm pair burdens was partially explained by the reduced fecundity of females from sexually biased infections. As eggs accumulated in the liver and portal hypertension increased, the number of injected microspheres escaping from the hepatic portal system rose in rank order of diameter from 9 μm through 15 μm and 25 μm to 50 μm. There was a strong correlation between the numbers of parasite eggs in the lungs and injected microspheres recovered from the lungs. The pattern of detection of microspheres in the lungs indicated a progressive increase in diameter of intra-hepatic porta-systemic connexions, followed by development of large-bore extra-hepatic collateral vessels. An accurate temporal profile of the pathological state of the host and the extent of collateral vessel formation was obtained. Injection of 141Ce-labelled microspheres demonstrated that the arterial supply to all organs of the body was affected by alterations in the micro-vasculature of the liver and lungs.

Pine wilt is the most serious disease of native pines in Japan and potentially the most important nematode disease of conifers in the world. The pinewood nematode Bursaphelenchus xylophilus was found to be the causal agent. Difficulties arose with respect to the precise identity of some isolates of B. xylophilus and of similar species B. mucronatus and B. fraudulentus. Restriction enzyme analyses of repetitive DNA revealed bands specific for the species B. xylophilus, B. mucronatus and B. fraudulentus. Hybridization patterns obtained with unc-22 gene of C. elegans, clearly identified B. xylophilus, B. mucronatus and B. fraudulentus as well as the different geographic isolates of these species. Furthermore, it is possible to define the phylogenetic relationships between the different populations constituting the ‘pine wood nematode’ complex.

Four new species of myxosporidian parasites are described from marine fishes from Plymouth: Sinuolinea rebae n.sp., from the urinary bladder of Solea solea; Leptotheca vikrami n.sp., from the gall bladder of Zeus faber; Zschokkella russelli n.sp., from the gall bladder of Gaidropsarus tricirratus and Ciliata mustela; and Zschokkella sturionis n.sp., from the gall bladder of Acipenser sturio.

The various terms used in describing the spore of Myxosporidia are reviewed and revised.

A new classification is proposed of the order Myxosporidia.

I wish to thank the Director of the Marine Biological Association's Laboratory for all the facilities he has enabled me to enjoy during my stay at Plymouth, by dedicating a new species of Zschokkella to him. As a mark of gratitude, and with a deep sense of respect to His Highness the Maharaja of Narsinghgarh State, C.I., I take the liberty of dedicating to him the new species of Leptotheca I have discovered. But for his encouragement and help in the early stages of my career it would have been impossible for me to apply for the India State Scholarship which I now hold. I take this opportunity of expressing my thanks to Miss N. G. Sproston for her unfailing help and encouragement during the present studies and am very grateful to her for going through the manuscript critically.

This work was carried out with a strain of trypanosomes isolated by inoculation into domestic rabbits of fleas naturally infected from wild rabbits in Hertfordshire.

The course of infection was studied in experimentally infected normal and splenectomized rabbits, in which the incubation period varied from 5 to 12 days. After recovery from the infection, rabbits became immune to re-infection. Immune rabbit serum had no protective action against Trypanosoma lewisi.

Attempts to infect other animals with the rabbit trypanosome produced negative results.

Young rabbits proved to be more susceptible to infection with this trypanosome, and their parasitaemia lasted longer than in adult rabbits, but the parasite was found to be non-pathogenic to these animals.

It was demonstrated that this trypanosome multiplies in the spleen, where the stages of division are confined to the capillaries. Multiplication follows the inoculation of metacyclic trypanosomes from infected fleas or blood forms. It proceeds by division of the nucleus and kinetoplast into four, followed by fission of the cytoplasm, giving rise to four small daughter individuals. These are transformed into trypanosome forms, which are shed into the peripheral blood, where they change into the typical adult trypanosomes.

The development in the intermediate host takes place in the gut of the rabbit flea, Spilopsyllus cuniculi, with the production of metacyclic trypanosomes in the hind-gut (posterior station).

The nomenclature and synonymy of the rabbit trypanosome are discussed, and it is concluded that its valid name is Trypanosoma nabiasi Railliet, 1895.

The influence of various agents on the adhesion of endospores of Pasteuria penetrans to the nematode Meloidogyne javanica was studied. Similarly, but to a lesser degree, we have also studied the adhesion of conidia of the fungus Dilophospora alopecuri and the coryneform bacterium Clavibacter sp. (syn. Corynebacterium rathayi) to the nematode Anguina agrostis (syn. A. funesta). Reduction in the degree of both spore and conidial attachment following their pre-treatment with periodate and the presence of PAS staining material on spores, conidia and bacteria implicated carbohydrate in these interactions. Tests involving both unbound and FITC-bound lectins demonstrated that wheat germ agglutinin (WGA) can inhibit the degree of attachment of P. penetrans to M. javanica and that this inhibition can be overcome by pre-treatment of the lectin with N, N′-diacetyl chitobiose. Endospores of P. penetrans, amphid and buccal secretions of 2nd-stage larvae of M. javanica and the cuticle and excretory pore secretions of 2nd-stage dauer larvae of A. agrostis bound WGA, indicating that accessible N-acetyl-D-glucosamine residues are present on these structures. Endospores of P. penetrans also bound Con A, indicating the presence of accessible α-D-glucose/α-D-mannose residues on their surface.