Optimising the hygiene of a liquid feeding system to improve the quality of liquid feed for pigs.

Poor feeding system hygiene may contribute to uncontrolled spontaneous fermentation in liquid pig feed and its associated undesirable effects. This study aimed to determine the effects of an intensive sanitisation programme in a grow-finisher liquid feeding system by monitoring microbiological and physico-chemical parameters of liquid feed and microbial colonisation of the feeding system surfaces. The sanitisation programme involved a combination of physical and chemical cleaning between batches of grow-finisher pigs, combined with nightly rinsing of the system with an organic acid blend. Improved hygiene of the internal surfaces of the mixing tank and feed pipeline, particularly until week 5 post-cleaning, was evidenced by reduced counts of lactic acid bacteria, total aerobes, Enterobacteriaceae, yeasts and moulds and decreased adenosine triphosphate concentrations. Enterobacteriaceae and moulds remained undetectable on pipeline surfaces for 10 weeks. Scanning electron microscopy of the feed pipelines confirmed these findings. Conversely, the impact on liquid feed microbiology was minimal and short-lived. However, acetic acid, ethanol and biogenic amine concentrations decreased in the feed post-cleaning and no gross energy losses were observed. Therefore, by controlling surface microbial communities on liquid feeding systems via implementation of the sanitisation programme developed in the current study, on-farm liquid feed quality should be improved.

Moderate genetic drift is driven by extreme recruitment events in the invasive mollusk Crepidula fornicata.

Effective population size (Ne) is a measure of genetic drift and is thus a central parameter in evolution, conservation genetics and invasion biology. Interestingly, in native marine species, Ne is typically several orders of magnitude lower than the census size. This pattern has often been explained by high fecundity, variation in reproductive success and pronounced early mortality, resulting in genetic drift across generations. Data documenting genetic drift and/or Ne in marine invasive species are, however, still scarce. We examined the importance of genetic drift in the invasive species Crepidula fornicata by genotyping 681 juveniles sampled during each annual recruitment peak over nine consecutive years in the Bay of Morlaix (Brittany, France). Observed variations in genetic diversity were partially explained by variation in recruitment intensity. In addition, substantial temporal genetic differentiation was documented (that is, genetic drift), and was attributed to nonrandom variance in the reproductive success of different breeding groups across years in the study species. Using a set of single-sample and temporal estimators for Ne, we estimated Ne to be three or four orders of magnitude smaller than the census size (Nc). On one hand, this reduction in Ne relative to Nc appeared congruent with, although slight higher than, values commonly observed in native marine species. Particular life-history traits of this invasive species may play an important role in buffering genetic drift. On the other hand, Ne still remained far below Nc, hence, possibly reducing the efficiency of selection effects.

Can c-myc amplification reliably discriminate postradiation from primary angiosarcoma of the breast?

PURPOSE: Breast angiosarcomas are rare vascular malignancies that arise secondary to irradiation or de novo as primary tumours. The aim of this study is to know whether c-myc amplification can reliably discriminate these two entities. MATERIEL AND METHODS: Forty-seven patients treated for breast angiosarcomas were studied. Thirty-two patients were diagnosed with postradiation angiosarcomas after breast cancer treatment and 15 patients with primary angiosarcomas. Interphase fluorescence in situ hybridization (FISH) was performed by hybridization of probes covering C-MYC (chromosome 8q24.21) and CEP8 on tissue sections. RESULTS: Amplification (5- to 20-fold) of the c-myc oncogene was found in all breast radiation-induced angiosarcomas (32 tumours) but in none of the 15 primary angiosarcomas except one (7%). CONCLUSION: This study reinforces that there are true pathogenetic differences between the two types of breast angiosarcomas which are morphologically indistinguishable. These data point the pathways preferentially involved in the pathogenesis of post radiation angiosarcomas of the breast and may provide the basis for an additional targeted therapy.

Towards a better understanding of biomarker response in field survey: a case study in eight populations of zebra mussels.

In order to provide reliable information about responsiveness of biomarkers during environmental monitoring, there is a need to improve the understanding of inter-population differences. The present study focused on eight populations of zebra mussels and aimed to describe how variable are biomarkers in different sampling locations. Biomarkers were investigated and summarised through the Integrated Biomarker Response (IBR index). Inter-site differences in IBR index were analysed through comparisons with morphological data, proteomic profiles and genetic background of the studied populations. We found that the IBR index was a good tool to inform about the status of sites. It revealed higher stress in more polluted sites than in cleaner ones. It was neither correlated to proteomic profiles nor to genetic background, suggesting a stronger influence of environment than genes. Meanwhile, morphological traits were related to both environment and genetic background influence. Together these results attest the benefit of using biological tools to better illustrate the status of a population and highlight the need of consider inter-population difference in their baselines.

PCR survey of 50 introns in animals: cross-amplification of homologous EPIC loci in eight non-bilaterian, protostome and deuterostome phyla.

Exon Primed Intron Crossing (EPIC) markers provide molecular tools that are susceptible to be variable within species while remaining amplifiable by PCR using potentially universal primers. In this study we tested the possibility of obtaining PCR products from 50 EPIC markers on 23 species belonging to seven different phyla (Porifera, Cnidaria, Arthropoda, Nematoda, Mollusca, Annelida, Echinodermata) using 70 new primer pairs. A previous study had identified and tested those loci in a dozen species, including another phylum, Urochordata (Chenuil et al., 2010). Results were contrasted among species. The best results were achieved with the oyster (Mollusca) where 28 loci provided amplicons susceptible to contain an intron according to their size. This was however not the case with the other mollusk Crepidula fornicata, which seems to have undergone a reduction in intron number or intron size. In the Porifera, 13 loci appeared susceptible to contain an intron, a surprisingly high number for this phylum considering its phylogenetic distance with genomic data used to design the primers. For two cnidarian species, numerous loci (24) were obtained. Ecdysozoan phyla (arthropods and nematodes) proved less successful than others as expected considering reports of their rapid rate of genome evolution and the worst results were obtained for several arthropods. Some general patterns among phyla arose, and we discuss how the results of this EPIC survey may give new insights into genome evolution of the study species. This work confirms that this set of EPIC loci provides an easy-to-use toolbox to identify genetic markers potentially useful for population genetics, phylogeography or phylogenetic studies for a large panel of metazoan species. We then argue that obtaining diploid sequence genotypes for these loci became simple and affordable owing to Next-Generation Sequencing development. Species surveyed in this study belong to several genera (Acanthaster, Alvinocaris, Aplysina, Aurelia, Crepidula, Eunicella, Hediste, Hemimysis, Litoditis, Lophelia, Mesopodopsis, Mya, Ophiocten, Ophioderma, Ostrea, Pelagia, Platynereis, Rhizostoma, Rimicaris), two of them, belonging to the family Vesicomydae and Eunicidae, could not be determined at the genus level.

Comparative phylogeography among hydrothermal vent species along the East Pacific Rise reveals vicariant processes and population expansion in the South.

The use of sequence polymorphism from individual mitochondrial genes to infer past demography has recently proved controversial because of the recurrence of selective sweeps acting over genes and the need for unlinked multilocus data sets. However, comparative analyses using several species for one gene and/or multiple genes for one species can serve as a test for potential selective effects and clarify our understanding of historical demographic effects. This study compares nucleotide polymorphisms in mitochondrial cytochrome oxidase I across seven deep-sea hydrothermal vent species that live along the volcanically active East Pacific Rise. Approximate Bayesian Computation (ABC) method, developed to trace shared vicariant events across species pairs, indicates the occurrence of two across species divergence times, and suggests that the present geographical patterns of genetic differentiation may be explained by two periods of significant population isolation. The oldest period dates back 11.6 Ma and is associated with the vent limpet Lepetodrilus elevatus, while the most recent period of isolation is 1.3 Ma, which apparently affected all species examined and coincides with a transition zone across the equator. Moreover, significant negative Tajima's D and star-like networks were observed for all southern lineages, suggesting that these lineages experienced a concomitant demographic and geographical expansion about 100 000-300 000 generations ago. This expansion may have initiated from a wave of range expansions during the secondary colonization of new sites along the Southern East Pacific Rise (founder effects below the equator) or recurrent bottleneck events because of the increase of eruptive phases associated with the higher spreading rates of the ridge in this region.

Fine- and regional-scale genetic structure of the exotic ascidian Styela clava (Tunicata) in southwest England, 50 years after its introduction.

Styela clava, an ascidian native to the northwest Pacific, was first recorded in the Atlantic at Plymouth, southwest England, in 1953. It now ranges in the northeast Atlantic from Portugal to northern Denmark, and has colonized the east coast of North America. Within the region of first introduction, we aimed to characterize current genetic diversity in the species, elucidate the respective roles of human-aided vs. natural dispersal, and assess the extent of larval dispersal by looking for genetic differentiation at very small scales. Eight sites, mostly marinas, were studied along c. 200 km of coast in southwest England encompassing Plymouth. Five microsatellite loci were genotyped in 303 individuals to analyse gene flow at regional (among sites) and fine (within sites) scales. F-statistics and assignment tests were used to investigate regional genetic structure. At the fine scale, deviation from mutation-drift equilibrium was tested, and isolation by distance and genetic clustering analyses were undertaken. Significant genetic differentiation existed between sites, unrelated to geographical separation; migration between geographically distant marinas was inferred, highlighting the likely importance of human-mediated dispersal in range expansion and occupancy by S. clava. Fine-scale population structure was present within at least four sites, which may be explained by the limited dispersal ability of this ascidian and recruitment from differentiated pools of larvae. Populations in enclosed marinas had higher self-recruitment rates than those in open sites. Some marinas might therefore function as reservoirs of propagules for subsequent spread, whereas others might be sinks for migrants.

PERMANENT GENETIC RESOURCES: Isolation of microsatellite loci from the kelp, Saccorhiza polyschides (Heterokontophyta, incertae sedis).

Kelps are ecologically important seaweeds that dominate the subtidal zones of rocky coasts. In Northern Europe, Saccorhiza polyschides is a pioneer species suspected of outcompeting the harvested kelp, Laminaria digitata. To examine how the process of species competition affects species distribution and genetic diversity in coastal environments, we developed 10 polymorphic microsatellite markers for S. polyschides using an enriched library (microsatellites are already available for L. digitata). These loci showed from three to 24 alleles with heterozygosities ranging from 0.36 to 0.92. This polymorphism is high enough for fine-scale population analyses including assignment tests to determine the origin of recruits.

Comparative effectiveness of sugar beet microsatellite markers isolated from genomic libraries and GenBank ESTs to map the sugar beet genome.

Sugar beet (Beta vulgaris) is an important root crop for sucrose production. A study was conducted to find a new abundant source of microsatellite (SSR) markers in order to develop marker assistance for breeding. Different sources of existing microsatellites were used and new ones were developed to compare their efficiency to reveal diversity in mapping population and mapping coverage. Forty-one microsatellite markers were isolated from a B. vulgaris ssp maritima genomic library and 201 SSRs were extracted from a B. vulgaris ssp vulgaris library. Data mining was applied on GenBank B. vulgaris expressed sequence tags (ESTs), 803 EST-SSRs were identified over 19,709 ESTs. Characteristics, polymorphism and cross-species transferability of these microsatellites were compared. Based on these markers, a high density genetic map was constructed using 92 F(2) individuals from a cross between a sugar and a table beet. The map contains 284 markers, spans over 555 cM and covers the nine chromosomes of the species with an average markers density of one marker every 2.2 cM. A set of markers for assignation to the nine chromosomes of sugar beet is provided.

Gregariousness and protandry promote reproductive insurance in the invasive gastropod Crepidula fornicata: evidence from assignment of larval paternity.

According to the size-advantage hypothesis, protandric sequential hermaphroditism is expected when the increase in reproductive success with age or size is small for males but large for females. Interestingly, some protandrous molluscs have developed gregarious strategies that might enhance male reproductive success but at the cost of intraspecific competition. The gastropod Crepidula fornicata, a European invading species, is ideal for investigating mating patterns in a sequential hermaphrodite in relation to grouping behaviour because individuals of different size (age) live in perennial stacks, fertilization is internal and embryos are brooded. Paternity analyses were undertaken in stacks sampled in three close and recently invaded sites in Brittany, France. Paternity assignment of 239 larvae, sampled from a set of 18 brooding females and carried out using five microsatellite loci, revealed that 92% of the crosses occurred between individuals located in the same stack. These stacks thus function as independent mating groups in which individuals may reproduce consecutively as male and female over a short time period, a pattern explained by sperm storage capacity. Gregariousness and sex reversal are promoting reproductive insurance in this species. In addition, females are usually fertilized by several males (78% of the broods were multiply sired) occupying any position within the stack, a result reinforcing the hypothesis of sperm competition. Our study pointed out that mating behaviours and patterns of gender allocation varied in concert across sites suggesting that multiple paternities might enhance sex reversal depending on sperm competition intensity.

Comparative phylogeography of two coastal polychaete tubeworms in the Northeast Atlantic supports shared history and vicariant events.

The historic processes which have led to the present-day patterns of genetic structure in the marine coastal fauna of the Northeast Atlantic are still poorly understood. While tectonic uplifts and changes in sea level may have caused large-scale vicariance, warmer conditions during glacial maxima may have allowed pockets of diversity to persist to a much wider extent than in the Northwestern Atlantic. The large-scale geographic distribution of deeply divergent lineages of the coastal polychaete tubeworms Pectinaria koreni (two clades) and Owenia fusiformis (three clades) were compared using a fragment of the mitochondrial cytochrome oxidase I gene (mtCOI). All lineages were present along the biogeographic transition zone on the north coast of Brittany (France) and we found evidence pointing towards congruence in the timing of cladogenic events between Pectinaria sp. (P. auricoma/P. belgica and P. koreni) and Owenia sp., suggesting a shared history of vicariant events. More conserved 16SrRNA sequences obtained from four species of Pectinariidae together with mtCOI sequences of P. koreni seem consistent with an initial establishment of pectinariids in the north, and a southward colonization of the Northeast Atlantic. Phylogeographic patterns in O. fusiformis were also consistent with a north/south pattern of lineage splitting and congruent levels of divergence were detected between lineages of both species. We observed signatures of both persistence in small northern glacial refugia, and of northwards range expansion from regions situated closer to the Mediterranean. However, whether the recolonization of the Northeast Atlantic by both species actually reflects separate interglacial periods is unclear with regards to the lack of molecular clock calibration in coastal polychaete species.

Multiple introductions promote range expansion of the mollusc Cyclope neritea (Nassariidae) in France: evidence from mitochondrial sequence data.

Since the 1970s, the nassariid gastropod Cyclope neritea has been extending its range north along the French Atlantic coasts from the Iberian Peninsula. This may be due to natural spread because of the recent warming of the northeastern Atlantic. However, human-mediated introductions related to shellfish culture may also be a probable explanation for this sudden range expansion. To examine these two hypotheses, we carried out a comprehensive study based on mitochondrial gene sequences (cytochrome oxidase I) of the five recently colonized French bays as well as 14 populations located in the recognized native range of the species. From a total of 594 individuals, we observed 29 haplotypes to split into three divergent clades. In the native range, we observed a low molecular diversity, strong genetic structure and agreement between geography and gene genealogies. Along the French coasts, we observed the opposite: high genetic diversity and low genetic structure. Our results show that recurrent human-mediated introductions from several geographical areas in the native range may be a source for the French Atlantic populations. However, despite the low dispersal ability of C. neritea, the isolation-by-distance pattern in France suggested that this gastropod may have been present (although unnoticed) on the French Atlantic coasts before the 1970s. As C. neritea shows characteristics of a cryptogenic species, the classification of Atlantic populations as either native or introduced is not straightforward. Cryptogenic species should be studied further to determine the status of new populations close to their recognized native range.

Sharp genetic break between Atlantic and English Channel populations of the polychaete Pectinaria koreni, along the North coast of France.

This study uses enzymatic and mitochondrial genes to infer the relative importance of historical processes and contemporary hydrodynamic features on the observed patterns of genetic structure in subdivided populations of Pectinaria koreni (Polychaeta: Pectinariidae) along the coasts of Brittany and the English Channel. Nucleotide sequence variation of a 603-bp fragment of the mtDNA cytochrome oxidase subunit I gene revealed a surprisingly deep phylogeographic break of about 16% divergence separating the Brittany and Channel populations, which coincides with a biogeographic boundary along the western coast of Brittany. Deep sequence divergence with fixed haplotype differences and the inversion of allele frequencies at two enzyme loci suggests the occurrence of potential cryptic or sibling species of P. koreni. The two clades showed opposite features. Channel populations exhibited bimodal match-mismatch curves due to two highly divergent haplotypes occurring at high frequencies and no overall heterozygote deficiencies at enzyme loci, suggesting respectively, a historic secondary contact between two differentiated populations followed by contemporary panmixia. On the contrary, Brittany populations displayed unimodal curves with low nucleotide diversity and highly significant heterozygote deficiencies, probably reminiscent of a recent population expansion and recolonisation of Brittany with contemporary admixture of divergent populations.

Evidence for gene flow via seed dispersal from crop to wild relatives in Beta vulgaris (Chenopodiaceae): consequences for the release of genetically modified crop species with weedy lineages.

Gene flow and introgression from cultivated to wild plant populations have important evolutionary and ecological consequences and require detailed investigations for risk assessments of transgene escape into natural ecosystems. Sugar beets (Beta vulgaris ssp. vulgaris) are of particular concern because: (i) they are cross-compatible with their wild relatives (the sea beet, B. vulgaris ssp. maritima); (ii) crop-to-wild gene flow is likely to occur via weedy lineages resulting from hybridization events and locally infesting fields. Using a chloroplastic marker and a set of nuclear microsatellite loci, the occurrence of crop-to-wild gene flow was investigated in the French sugar beet production area within a 'contact-zone' in between coastal wild populations and sugar beet fields. The results did not reveal large pollen dispersal from weed to wild beets. However, several pieces of evidence clearly show an escape of weedy lineages from fields via seed flow. Since most studies involving the assessment of transgene escape from crops to wild outcrossing relatives generally focused only on pollen dispersal, this last result was unexpected: it points out the key role of a long-lived seed bank and highlights support for transgene escape via man-mediated long-distance dispersal events.

Introgression patterns in the mosaic hybrid zone between Mytilus edulis and M. galloprovincialis.

Hybrid zones are fascinating systems to investigate the structure of genetic barriers. Marine hybrid zones deserve more investigation because of the generally high dispersion potential of planktonic larvae which allows migration on scales unrivalled by terrestrial species. Here we analyse the genetic structure of the mosaic hybrid zone between the marine mussels Mytilus edulis and M. galloprovincialis, using three length-polymorphic PCR loci as neutral and diagnostic markers on 32 samples along the Atlantic coast of Europe. Instead of a single genetic gradient from M. galloprovincialis on the Iberian Peninsula to M. edulis populations in the North Sea, three successive transitions were observed in France. From South to North, the frequency of alleles typical of M. galloprovincialis first decreases in the southern Bay of Biscay, remains low in Charente, then increases in South Brittany, remains high in most of Brittany, and finally decreases again in South Normandy. The two enclosed patches observed in the midst of the mosaic hybrid zone in Charente and Brittany, although predominantly M. edulis-like and M. galloprovincialis-like, respectively, are genetically original in two respects. First, considering only the various alleles typical of one species, the patches show differentiated frequencies compared to the reference external populations. Second, each patch is partly introgressed by alleles of the other species. When introgression is taken into account, linkage disequilibria appear close to their maximum possible values, indicating a strong genetic barrier within all transition zones. Some pre- or postzygotic isolation mechanisms (habitat specialization, spawning asynchrony, assortative fertilization and hybrid depression) have been documented in previous studies, although their relative importance remains to be evaluated. We also provided evidence for a recent migratory 'short-cut' connecting M. edulis-like populations of the Charente patch to an external M. edulis population in Normandy and thought to reflect artificial transfer of spat for aquaculture.

Dynamics and responsiveness of T-lymphocytes in secondary lymphoid organs of rabbits developing immunity to Eimeria intestinalis.

Primary infection with Eimeria intestinalis confers very effective immunity against further infections in rabbits. This study was designed to determine the onset of the immune response in primary-infected rabbits and to characterise the immune status of protected rabbits. Variations in kinetics of CD4+ and CD8+ T-cell subpopulations were followed after primary infection at the intestinal sites of penetration (duodenum) and development (ileum), in mesenteric lymph nodes (MLN) and in the spleen. The response against the parasite was measured by specific lymphocyte proliferation in the spleen and MLN and by determining specific IgG titres in serum. The mucosal immune response was strong after primary infection and was characterised by (i) transient increase in the percentages of intestinal CD4+ lymphocytes and MLN CD8+ lymphocytes 14 days PI and (ii) strong increase in the percentages of intestinal CD8+ lymphocytes from 14 days PI persisting throughout further infections. Extensive infiltration of the lamina propria with CD8+ lymphocytes was observed 14 days PI. The specific proliferative response started between 7 and 14 days PI in MLN but remained undetectable in spleens for up to 21 days, in contrast to "immunised" rabbits. The fact that systemic immune responses were low after primary infection, in contrast to indicators of mucosal immune responsiveness, suggests that protection of rabbits against E. intestinalis infection is due to an effective mucosal immune response, and that systemic responses that increase after successive infections are only reflections of repeated encounters with parasite antigens.

Crop-weed interactions in the Beta vulgaris complex at a local scale: allelic diversity and gene flow within sugar beet fields.

Crop-wild hybrids and weed beets are the main source of agronomic concern for sugar beet production all over Europe. In order to understand the dynamics of crop-wild interactions and the evolution of weediness in Beta vulgaris, we investigated genetic features of bolting individuals occurring at a local scale, i.e. within two sugar beet fields of the French northern area of sugar beet production. By analysing ploidy level, mitochondrial DNA and microsatellite polymorphism, the genetic diversity and the genetic relationships among three different classes of individuals (variety, in-row and out-row weed-beets) from a given field were examined. Such genetic analyses provide a unique opportunity to obtain evidence for the weeds origin and the evolutionary hypotheses previously stated. All the individuals shared in common the Svulg mitochondrial haplotype, and thus a common maternal origin. Conversely, the large genetic diversity at microsatellite loci highlighted the large diversity of the pollinator plants (cultivated and wild plants) during the-seed production process, as well as during the further evolution of weed beets in the sugar production area.

Diversity and genetic structure in populations of Pseudotsuga menziesii (Pinaceae) at chloroplast microsatellite loci.

Genetic variation was compared between uniparentally-inherited (chloroplast simple sequence repeats, cpSSRs) vs. biparentally-inherited (isozyme and random amplified polymorphic DNA, RAPD) genetic markers in Douglas-fir (Pseudotsuga mensiezii) from British Columbia. Three-hundred twenty-three individuals from 11 populations were assayed. In Douglas-fir, the cpSSR primer sites were well-conserved relative to Pinus thunbergii (11 of 17 loci clearly amplified), but only 3 loci were appreciably polymorphic. At these cpSSR loci, we found an unexpectedly low level of polymorphism within populations, and no genetic differentiation among populations. By contrast, the nuclear markers showed variation typical of conifers, with significant among-population differentiation. This difference is likely the outcome of both historical factors and high pollen dispersal.

The spatial structure of sexual and cytonuclear polymorphism in the gynodioecious Beta vulgaris ssp. maritima: I/ at a local scale.

We have analyzed the spatial distribution of the sex phenotypes and of mitochondrial, chloroplast, and nuclear markers within two gynodioecious populations of Beta vulgaris ssp. maritima. Within both populations, sexual phenotype variation is controlled mainly by the cytoplasmic genotype, although in one study population a joint polymorphism of cytonuclear factors is clearly involved. In spite of contrasts in the ecology (mainly due to different habitats), a clear common feature in both populations is the highly patchy distribution of cytoplasmic haplotypes, contrasting with the wide distribution of nuclear diversity. This high contrast between cytoplasmic vs. nuclear spatial structure may have important consequences for the maintenance of gynodioecy. It provides opportunities for differential selection since nuclear restorer alleles are expected to be selected for in the presence of their specific cytoplasmic male sterile (CMS) type, but to be neutral (or selected against if there is a cost of restoration) in the absence of their CMS type. Selective processes in such a cytonuclear landscape may explain the polymorphism we observed at restorer loci for two CMS types.

Tissues and cells involved in the invasion of the rabbit intestinal tract by sporozoites of Eimeria coecicola.

This study was designed to identify an extra-intestinal route of migration of Eimeria coecicola sporozoites and the types of cell harbouring the parasite during the invasion of the intestine. The presence of E. coecicola in blood, spleen and mesenteric lymph nodes of infected donor rabbits was demonstrated by immunohistology on donor organs and measurement of oocyst excretion by coccidia-free recipient rabbits injected with whole-cell suspensions prepared from donor tissues. Two types of donor lymphocyte, B (IgM+) and T (CD5+), were labelled using a two-colour immunofluorescence-labelling technique and separated with a cell-sorter (FACStar(Plus)). The presence of parasites in the sorted cells was assessed by direct examination and by using the same in vivo test after intravenous injection of IgM+ B or CD5+ T lymphocytes collected from donors at different times after inoculation. This test provided evidence that the parasites were alive and still infectious within the sorted lymphocytes. It was demonstrated that both B and T lymphocytes were infected.