The red poultry mite, Dermanyssus gallinae, is one of the most economically deleterious ecto-parasites of layer hens worldwide. D. gallinae is difficult to eliminate from infested poultry farms, and even to study, because it resides on the host only during the bloodmeal at night, and hides in the crevices of poultry houses during the day. Here, the life-cycle of D. gallinae was reproduced entirely in vitro. Mites were incubated in a glass pipette at 30 °C, 60–95% relative humidity and total darkness. A feeding apparatus, composed of a membrane, reservoir and blood was fitted on the pipette during bloodmeals. We tested feeding rates on blood mixed with 1 of 3 anti-coagulants (EDTA, heparin and trisodium citrate) at different concentrations, and biological and artificial membranes. The best engorgement and survival rates for all 3 haematophagous life-stages of the parasite were observed in 1-day-old chick membranes and heparinized (0.02 mmol/ml) blood. We then describe the steps in developing a complete self-sustaining in vitro life-cycle. A colony of mites was maintained in vitro for 7 generations. Losses in the first generation were heavy, but survival had multiplied 5-fold by the fifth generation. We hypothesize that heavy mortality rates during the first life-cycle correspond to selective pressure: only the mites which fed and survived in vitro were able to reproduce.

The global increase of the human parasite, the common bed bug Cimex lectularius, calls for specific pest control target sites. The bed bug is also a model species for sexual conflict theory which suggests that seminal fluids may be highly diverse. The species has a highly unusual sperm biology and seminal proteins may have unique functions. One-dimensional PAGE gels showed 40–50% band sharing between C. lectularius and another cimicid species, Afrocimex constrictus. However, adult, sexually rested C. lectularius males were found to store 5–7 μg of seminal protein and with only 60 μg of protein we obtained informative 2-D PAGE gels. These showed 79% shared protein spots between 2 laboratory populations, and more than half of the shared protein spots were detected in the mated female. Further analysis using liquid chromatography electrospray ionization tandem mass spectrometry revealed that 26·5% of the proteins had matches among arthropods in databases and 14·5% matched Drosophila proteins. These included ubiquitous proteins but also those more closely associated with reproduction such as moj 29, ubiquitin, the stress-related elongation factor EF-1alpha, a protein disulfide isomerase and an antioxidant, Peroxiredoxin 6.

The pathogenic Theileria species Theileria parva and T. annulata infect bovine leukocytes and erythrocytes causing acute, often fatal lymphoproliferative diseases in cattle. The parasites are of interest not only because of their economic importance as pathogens, but also because of their unique ability to transform the leukocytes they infect. The latter property allows parasitized leukocytes to be cultured as continuously growing cell lines in vitro, thus providing an amenable in vitro system to study the parasite/host cell relationship and parasite-specific cellular immune responses. This paper summarizes important advances in knowledge of the immunobiology of these parasites over the last 40 years, focusing particularly on areas of relevance to vaccination.

The variability inherent in different isolates of Fasciola hepatica has been evident from reports in the literature but to date there has been no systematic examination of the relationship between these differences and the fecundity of the parasite. In this study we have attempted to remedy this situation by comparing the relative efficiencies with which 2 well-characterized isolates of the liver fluke (Oberon and Fairhurst) progress through both their definitive and intermediate hosts. We did not observe a reduction in fitness in the Oberon isolate which has been reported to be triclabendazole-resistant, compared to the triclabendazole-susceptible Fairhurst isolate, but considerable inter- and intra-isolate variability at different life-cycle stages was recorded. Thus the Oberon isolate gave 4-fold the number of cercariae when 100 snails were each challenged with a single miracidium and was more successful in establishing productive infections in rats. Fairhurst metacercariae excysted at a higher rate than those from the Oberon isolate and Fairhurst flukes produced 4-fold more eggs. The extent of the intra- and inter-isolate variability revealed in this work will provide a basis for the development of models of population dynamics aimed at predicting the response of the liver fluke to changing environmental conditions such as the use of anthelmintics or climatic change.

Due to their widespread distribution and virulence, protozoan species of the genus Perkinsus are especially worrisome parasites for shellfish farmers. In the present paper, we investigate the organization and the structural features of the nuclear ribosomal genes of Perkinsus atlanticus as well as the use of DNA sequence information from this region for phylogenetic analyses. This information has been useful, further, for the development of a diagnostic test based on the amplification by the polymerase chain reaction (PCR) technique. We have isolated a high-copy DNA sequence in this species, and, after its characterization, we have determined that it corresponds to the ribosomal RNA (rRNA) genes 28S–5S–18S and the intergenic spacers. By comparing the complete sequence of the 5S rRNA gene and a partial sequence of the 18S rRNA gene of P. atlanticus with the sequences of those genes in other Alveolates, we have found additional support for the hypothesis that Perkinsus is more closely related to species of Dinoflagellata than to species of Apicomplexa. The intergenic spacer sequence between the 5S and the 18S rRNA genes was used to design a pair of primers to be used as a PCR-based diagnostic test.The nucleotide sequence data reported in this paper are available in the EMBL database with the accession number AJ238400.The commercial use of the diagnostic test presented here is protected by a patent. This patent is pending upon the application number P9801511.

Gyrodactylus arcuatus-like monogeneans collected from the gills of 3 goby species, Pomatoschistus minutus, P. lozanoi and P. pictus, from the Voordelta Area (SW Netherlands) were compared on a morphometric basis with G. arcuatus from its type host, the three-spined stickleback, Gasterosteus aculeatus. Univariate and multivariate analysis was carried out on measurements of 17 characters of the opisthaptoral hard parts of 268 specimens. The contribution of host-dependent and seasonal factors to the observed morphological variation are discussed. Univariate statistics (ANOVA) were used to detect features that were useful in separating the gyrodactylids from the different host groups. Subsequent factor analysis and discriminant analysis, combining all variables, led to the separation of 3 distinct forms (possibly species) dependent on the host species harbouring them. G. arcuatus (sensu stricto) was only present on G. aculeatus, while P. minutus and P. lozanoi carried a common species, Gyrodactylus sp. 1, and P. pictus hosted Gyrodactylus sp. 2. Seasonal variation in size of the sclerities was demonstrated on the Gyrodactylus species from P. minutus and P. lozanoi, and should be taken into consideration when specimens are compared. The findings are discussed in view of the possibility of phylogenetic host specificity of the gyrodactylids occurring on goby species that live sympatrically in the Voordelta area.

An AFLP approach was established to investigate genetic diversity within Oesophagostomum bifurcum (order Strongylida) from human and non-human primates. Evaluation of different combinations of restriction enzymes (n=8) and primers (n=29) demonstrated that the use of HindIII/BglII digested templates and primers with the selective nucleotides +AG/+AC, respectively, was the most effective for the analysis of O. bifurcum DNA. A total of 63 O. bifurcum adults from human, Patas monkey, Mona monkey and Olive baboon hosts from different geographical regions in Ghana were subjected to analysis using this method. Cluster analysis revealed 4 genetically distinct groups, namely O. bifurcum from the Patas monkey (I), from the Mona monkey (II), from humans (III) and from the Olive baboon (IV). These findings were concordant with those achieved previously using RAPD analysis and supports population genetic substructuring within O. bifurcum according to host species. The results demonstrated the effectiveness of the present AFLP method for establishing genetic variation within O. bifurcum, and indicates its applicability to other parasitic nematodes of human and/or veterinary health importance.

Understanding the frequency distribution of parasites and parasite stages among hosts is essential for efficient experimental design and statistical analysis, and is also required for the development of sustainable methods of controlling infection. Nematodirus battus is one of the most important organisms that infect sheep but the distribution of parasites among hosts is unknown. An initial analysis indicated a high frequency of animals without N. battus and with zero egg counts, suggesting the possibility of a zero-inflated distribution. We developed a Bayesian analysis using Markov chain Monte Carlo methods to estimate the parameters of the zero-inflated negative binomial distribution. The analysis of 3000 simulated data sets indicated that this method out-performed the maximum likelihood procedure. Application of this technique to faecal egg counts from lambs in a commercial upland flock indicated that N. battus counts were indeed zero-inflated. Estimating the extent of zero-inflation is important for effective statistical analysis and for the accurate identification of genetically resistant animals.

Viable eggs produced weekly per infective stage was used as a measure of the reproductive success of Echinostoma friedi during the first 12 weeks of infection in hamsters. The weekly reproductive success was not constant during the experiment in relation to the egg output and the proportion of viable eggs produced. The egg release started during week 2 post-inoculation, attaining a maximum during week 3. A decline in egg output was observed from week 9. Viable eggs were only produced from week 3 post-inoculation and a maximum was attained at week 4 of the experiment. A decline in egg viability was observed from week 9. Considering together the egg output and the egg viability, the maximum weekly reproductive success was obtained during week 4 post-inoculation. The changes in the weekly reproductive success were not reflected in variations in worm numbers and body sizes during the course of the infection. The humoral immune response of golden hamsters during the infection with E. friedi was determined. Increases of IgG levels against somatic and excretory/secretory products of E. friedi were detected coinciding with the reduction in the reproductive success.

A striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4–5 kDa. Two different monoclonal antibodies directed against the 4–5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the low Mr antigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4–5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability between Toxoplasma gondii tachyzoites grown in vivo and in vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

The unique environment of the Mediterranean Sea makes fish stock assessment a major challenge. Stock identification of Mediterranean fisheries has been based mostly from data on biology, morphometrics, artificial tags, otolith shape and fish genetics, with less effort on the use of parasites as biomarkers. Here we use some case studies comparing Mediterranean vs Atlantic fish stocks in a multidisciplinary framework. The generalized Procrustes Rotation (PR) was used to assess the association between host genetics and larval Anisakis spp. datasets on demersal (hake) and pelagic (horse mackerel, swordfish) species. When discordant results emerged, they were due to the different features of the data. While fish population genetics can detect changes over an evolutionary timescale, providing indications on the cohesive action of gene flow, parasites are more suitable biomarkers when considering fish stocks over smaller temporal and spatial scales, hence giving information of fish movements over their lifespan. Future studies on the phylogeographic analysis of parasites suitable as biomarkers, and that of their fish host, performed on the same genes, will represent a further tool to be included in multidisciplinary studies on fish stock structure.

The monogenean Gyrodactylus salaris Malmberg, 1957 is an economically important parasite on Atlantic salmon whereas the morphologically very similar G. thymalli Žitňan, 1960 on grayling is considered harmless. Even molecular markers cannot unambiguously discriminate both species. The nuclear internal transcribed spacer (ITS) sequences are identical in both species, and although mitochondrial cytochrome oxidase I (COI) sequences show substantial variation, no support for monophyly of either species is found. Analysis of nucleotide sequences of the intergenic spacer (IGS) have, however, been interpreted as support for 2 species. Here, IGS and COI sequences from 81 G. salaris and G. thymalli specimens of 39 populations across the species' distribution range were determined. Mitochondrial diversity was not reflected in the nuclear marker. Since various 23 bp IGS repeat types usually differ by just one nucleotide and sequences primarily differ in the number and order of repeat types, alignments may be biased and arbitrary, impeding meaningful phylogenetic analyses. The hypothesis that parasites on rainbow trout represent hybrids of both species is rejected. The presence or absence of particular repeat types is not considered informative. We interpret the IGS data as support for G. salaris and G. thymalli being a single species.

Pasture plots in a warm coastal region were separately contaminated in the autumn, winter and spring with eggs of a coastal strain of Ostertagia ostertagi derived from dairy calves. At the same times, a parallel set of plots was contaminated by a strain transferred from within a beef cattle management system in a cooler tablelands region. A reciprocal transfer of the coastal strain was also made to the tablelands, where pasture plots were contaminated with eggs of both strains of O. ostertagi in winter. Successive groups of parasite-free calves were grazed on the plots for 10–14 days at 4-week intervals and then killed for worm counts 14 days after their removal from pasture. In both environments, irrespective of the season of pasture contamination, inhibition of development reached a maximum in spring and the two strains differed only in the proportion of the population inhibited, which was significantly greater for the table- lands strain. There was no strain difference for either numbers of infective larvae on pasture or total O. ostertagi numbers. This suggested that the difference between coastal and tablelands populations in the extent of inhibition is genetically determined. If an environmental stimulus was implicated in the spring occurrence of inhibition, the stimulus was provided equally in coastal and tablelands environments and it cannot be exposure to low temperatures.

Larvae of the monogeneans Pseudodiplorchis americanus and Neodiplorchis scaphiopodis develop within egg capsules retained in the uterus of the parent parasite. This study reveals adaptations for the storage and nutrition of infective stages which have no known precedent amongst other platyhelminths. The vitelline system is greatly reduced and appears to contribute little to embryo nutrition. An electron-dense ‘shell’ which encloses the newly formed embryos is subsequently replaced by a thin flexible sac composed of concentric layers of membranes which are derived from the uterus wall. Further membranes are added to this multi-laminate structure as the encapsulated embryos pass along the uterus, and this enables the sac to expand continuously as the larva inside it grows. The capsule lining extends into fine cytoplasmic processes which ‘plumb in’ to the larval tegument between ciliated cells. These connexions are packed with glycogen and appear to perform a placenta-like function. The unique adaptations correlate with the demands of the life-cycle. Large numbers of oncomiracidia (up to 300) can be packed, in membranous sacs, within the storage capacity of the uterus; direct transfer of nutrients from parent to offspring enables resources to be supplied over an extended period; and infective larvae can be maintained in readiness for an unpredictable opportunity for transmission.

Administration of the immunostimulants Corynebacterium parvum, Bacillus Calmette-Guérin (BCG) or Bordetella pertussis prior to, or at the same time as, challenge with Trypanosoma congolense significantly increased survival times in mice, both of trypano-susceptible (A/J) and trypano-resistant (C57Bl) strains. The increased survival time was associated with significant alterations in parasitaemia, which included lengthening of the pre-patent period, a delay in the time taken to reach the first peak of parasitaemia and a reduction in the level of parasitaemia. Similar results were obtained when these strains of mice were challenged with Trypanosoma brucei following pre-treatment with C. parvum. Thus, by the use of immunostimulants it was possible to reduce the susceptibility of mice to trypanosomiasis and the hope is that this can also be achieved with domestic livestock.

The whole life cycle of Paramphistomum microbothrium Fischoeder, 1901, found in cattle of Kenya, East Africa, has been established experimentally.

In laboratory conditions, the eggs hatched miracidia on the 14th to 16th day, if they were kept in water at a temperature of 26–28° C.

In a snail, Bulinus alluaudi (Dautzenberg), kept at the temperature of 18–20° C., the miracidium developed into a sporocyst, the elongated body of which, containing young rediae, reached a length of 3·6 mm. in about 2 weeks.

The first rediae which emerged from a sporocyst were observed on the 14th day, and on the 20th day the first generation rediae began to produce second-generation rediae. From the 28th day onward the first-generation parent rediae ceased to produce daughter rediae and began to develop cercariae only. This period of production of cercariae by the redia lasted about 30 days, and when the life of the first-generation rediae drew to its close, the old rediae developed a few daughter rediae again.

Cercariae began to emerge from the first-generation rediae 30 days after exposure of the snail to miracidia. The cercariae left the parent redia in a very immature state and further development occurred in the liver of the snail. The emergence of cercariae from the infected snail began on the 43rd day after exposure to miracidia. Shortly after emerging from the snail, the cercariae attached themselves to vegetation and encysted.

Development of rediae of the second, third, fourth and apparently more successive generations followed an identical course to that outlined for the rediae of the first generation. As a result the successive generations of rediae maintain the infection going in an intermediate host and the infected snails were continually shedding cercariae as long as they lived. In the laboratory experiments the life span of some of the infected snails exceeded a year.

In cattle infected experimentally P. microbothrium reached maturity and began passing out the eggs about a 100 days after the encysted cercariae were fed to the animals.

Total DNAs, isolated from a range of taeniid cestodes (Taenia solium, T. saginata, T. pisiformis, T. crassiceps, T. hydatigena, T. ovis, T. multiceps and T. taeniaeformis), have been subjected to restriction enzyme digestion, Southern transfer and hybridization analysis using cloned fragments of the ribosomal RNA gene of Schistosoma mansoni. Substantial inter-specific genetic differences have been revealed on the basis of characteristic hybridization patterns for each of the taeniid cestode species. Furthermore, a random genomic DNA library has been constructed in the vector plasmid pAT153 using DNA extracted from a pig isolate (Indian origin) of T. solium. A panel of taeniid cestode DNAs including DNA from Echinococcus granulosus, has been used in conjunction with hybridization and restriction enzyme analysis to identify in the library a single recombinant plasmid with a T. solium-specific insert (coded pTS10) and two recombinant plasmids with T. solium inserts having selective specificities for T. solium and T. ovis (coded pTS17) and T. solium, T. saginata, T. ovis and T. multiceps (coded pTS28). These recombinant plasmids and the cloned fragments of the ribosomal RNA gene of S. mansoni have been used in restriction endonuclease, Southern transfer and hybridization analysis to detect intra-specific genetic variation in cysticerci of T. solium from India, Mexico and Zimbabwe. In addition, pTS10 and pTS17 have been used in a simple dot-blot assay to distinguish T. solium from T. saginata.

We have examined several taxonomic features of a number of strains of Eimeria from many sources world wide. The strains were isolated on the basis of their small spherical (or sub-spherical) oocysts. From a study of mean oocyst dimensions, electrophoretic variation of enzymes, ability to develop in embryonated eggs, absence of gross lesions in heavily infected chickens, and cross-immunity, all the strains were found to belong to one species. For convenience, the parasites when isolated, were referred to as strains of E. mitis/mivati-type, but after characterization they were clearly found to be E. mitis. In contrast, a laboratory strain of E. mivati supplied to one of us (M.W.S.) was found to be a mixture of E. acervulina and E. mitis. Evidence from these and other studies supports the notion that E. mivati is a nomina dubia.

Acquired resistance to some nematode parasites can be suppressed by daily administration of cortisone acetate to the host. Cortisone treatment completely suppressed previously acquired resistance of mice to Trichinella spiralis (Coker, 1956) and suppressed, at least partially, the acquisition of resistance to T. spiralis by rats during initial infection (Markell & Lewis, 1957).

A previous report (Weinstein, 1955) suggested cortisone acetate treatment was less effective in the suppression of acquired resistance of rats to Nippostrongylus brasiliensis. Weinstein showed that daily treatment with 2 mg cortisone acetate throughout five immunizing infections and continued during the challenge infection increased the number of worms in the intestine and had a marked effect on the cellular response in the skin and lungs. However, there was no significant effect when daily cortisone treatment commenced only on the fifth day before the challenge infection. This result suggested that acquisition of immunity to N. brasiliensis was partially overcome by cortisone treatment, but the same level of drug treatment had no effect on immunity already acquired.

In the experiments reported here, previously acquired resistance to N. brasiliensis was suppressed completely and the initiation of immunity stopped, either completely or at a very early stage, by treatment with the cortisone derivatives prednisolone or betamethasone. The complete suspension of all manifestations of acquired resistance obtained by treatment with these drugs was used to investigate the fate of larvae migrating in an immune host.

The rats and strain of parasite, and the methods of handling them, have been described previously (Ogilvie, 1965).

Eimeria praecox produced less effect on body-weight gain and food consumption than E. acervulina. No deaths occurred and no weight loss followed infection with doses of oocysts up to 10. However, there was a depression of body-weight gain in birds infected with both species and changes in the permeability of the intestine were noted from as early as 3½ h after infection. This suggests that increased intestinal permeability is not the major factor in the greater pathogenicity of E. acervulina compared with E. praecox. Eimeria acervulina produced considerably greater effects on the host as evidenced by mortality, body weight gain and food consumption.

I wish to thank Mr B. J. Millard and Mr M. Shirley for technical assistance and Dr B. M. Freeman for help with the statistics in Table 2.