In vitro cultivation of Trypanosoma congolense bloodstream forms: State of the art and advances.

Animal African Trypanosomosis (AAT or Nagana) is a severe vector-borne disease caused by protozoan parasites belonging to the Trypanosomatidae family and is usually cyclically transmitted by blood-sucking tsetse flies. AAT remains a major problem in sub-Saharan Africa. Among the main AAT causative agents, Trypanosoma congolense (T. congolense or Tc) is one of the most important trypanosome species, in terms of economic and animal health impacts, infecting cattle and a wide range of animal hosts as well. To advance in AAT prevention and control, it is essential to better understand trypanosome biology and pathogenesis using bloodstream form (BSF) in vitro culture. The in vitro cultivation of T. congolense IL3000 BSF strain is already well established and widely used in research studies and drug activity assays. However, it may probably no longer truly reflect the reality of field trypanosome strains, due to decades of use and subsequent modifications. Here, we propose a novel culture protocol that supports the long-term in vitro growth of the animal-infective BSFs of three Savannah and Forest types of T. congolense strains, including T. congolense clone IL1180, which is not only a field strain but also a commonly-used reference strain in experimental animal assays. We established a homemade culture medium which made it possible to sustain T. congolense IL1180 growth from infected mouse blood for 18 days in axenic conditions. Moreover, we developed an efficient freezing/thawing system that allowed, for the first time, T. congolense IL1180 BSF growth within 30 days after thawing. Our results on T. congolense adaptation to in vitro culture are encouraging for future gene studies using new molecular tools or for new therapeutic drug assays.

Erratum to: Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

Unfortunately, the original version of this article [1] contained an error. Figure 1 in the original article, corresponded to the first coinertia analysis that was carried out with no data on the procyclin PE repeats for the T. brucei brucei strains. After including these data, the coinertia analysis was modified both in the directionality of the arrows in the Y Hyperspace and in the biplot generated by the interaction of the two coinertia axes. The modified coinertia analysis is included in Fig. 1.

Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

BACKGROUND: Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia. METHODS: Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software. RESULTS: We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain. CONCLUSION: Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.

Association studies in QTL regions linked to bovine trypanotolerance in a West African crossbred population.

African animal trypanosomosis is a parasitic blood disease transmitted by tsetse flies and is widespread in sub-Saharan Africa. West African taurine breeds have the ability, known as trypanotolerance, to limit parasitaemia and anaemia and remain productive in enzootic areas. Several quantitative trait loci (QTL) underlying traits related to trypanotolerance have been identified in an experimentally infected F(2) population resulting from a cross between taurine and zebu cattle. Although this information is highly valuable, the QTL remain to be confirmed in populations subjected to natural conditions of infection, and the corresponding regions need to be refined. In our study, 360 West African cattle were phenotyped for the packed cell volume control under natural conditions of infection in south-western Burkina Faso. Phenotypes were assessed by analysing data from previous cattle monitored over 2 years in an area enzootic for trypanosomosis. We further genotyped for 64 microsatellite markers mapping within four previously reported QTL on BTA02, BTA04, BTA07 and BTA13. These data enabled us to estimate the heritability of the phenotype using the kinship matrix between individuals computed from genotyping data. Thus, depending on the estimators considered and the method used, the heritability of anaemia control ranged from 0.09 to 0.22. Finally, an analysis of association identified an allele of the MNB42 marker on BTA04 as being strongly associated with anaemia control, and a candidate gene, INHBA, as being close to that marker.

Population genetic structure of wild and farmed rusa deer (Cervus timorensis russa) in New-Caledonia inferred from polymorphic microsatellite loci.

Historical records indicate that 12 rusa deer (Cervus timorensis russa) were introduced in New-Caledonia during the 1870s. We used eight polymorphic microsatellite DNA loci to assess the genetic differentiation and diversity of farmed and wild deer populations. Past genetic bottlenecks were detected in both sub-populations, although higher genetic diversity was maintained in farmed populations, probably due to the regular introduction of reproducers from wild populations and from other farms. The genetic structure of farmed and wild populations differed significantly. There was a significant isolation by distance for wild populations, whereas farmed populations were significantly differentiated between farms independently from their geographical proximity. Wild rusa deer consisted of small populations (with effective population sizes ranging between 7 and 19 individuals depending on the methods used), with a low parent-offspring dispersion range (0.20-2.02 km). Genetic tools and direct observations provided congruent estimates of dispersion and population sizes. We discuss the relevance of our results for management purposes.

Detection of selection signatures within candidate regions underlying trypanotolerance in outbred cattle populations.

Breeding indigenous African taurine cattle tolerant to trypanosomosis is a straightforward approach to control costs generated by this disease. A recent study identified quantitative trait loci (QTL) underlying trypanotolerance traits in experimental crosses between tolerant N'Dama and susceptible Boran zebu cattle. As trypanotolerance is thought to result from local adaptation of indigenous cattle breeds, we propose an alternative and complementary approach to study the genetic architecture of this trait, based on the identification of selection signatures within QTL or candidate genes. A panel of 92 microsatellite markers was genotyped on 509 cattle belonging to four West African trypanotolerant taurine breeds and 10 trypanosusceptible European or African cattle breeds. Some of these markers were located within previously identified QTL regions or candidate genes, while others were chosen in regions assumed to be neutral. A detailed analysis of the genetic structure of these different breeds was carried out to confirm a priori grouping of populations based on previous data. Tests based on the comparison of the observed heterozygosities and variances in microsatellite allelic size among trypanotolerant and trypanosusceptible breeds led to the identification of two significantly less variable microsatellite markers. BM4440, one of these two outlier loci, is located within the confidence interval of a previously described QTL underlying a trypanotolerance-related trait. Detection of selection signatures appears to be a straightforward approach for unravelling the molecular determinism of trypanosomosis pathogenesis. We expect that a whole genome approach will help confirm these results and achieve a higher resolving power.

First outbreak of Trypanosoma evansi in camels in metropolitan France.

The first outbreak of trypanosomosis caused by Trypanosoma evansi in camels in France was reported on a farm in the Aveyron Department. Five camels were imported from the Canary Islands to the farm in early July 2006, and trypanosomes were observed on a stained blood smear from one of them, which died in October. On further investigations, trypanosomes were observed in the blood of five camels, three of them indigenous to the farm and two that had been imported. On the basis of microscopical examination (morphological criteria and measurements) and serological results based on the card agglutination T evansi test and PCR typing, the parasites were identified as T evansi. After treatment with melarsomine, the infected camels rapidly became negative by parasitological tests and were negative two to four months later by serological tests. The parasite was probably transmitted by tabanids and Stomoxys calcitrans, which were abundant in July to September 2006. No parasites were observed in other animals on the farm or on neighbouring farms, but some of the sheep on these farms were positive by PCR or serology.

The extent of linkage disequilibrium in a large cattle population of western Africa and its consequences for association studies.

Several previous studies concluded that linkage disequilibrium (LD) in livestock populations from developed countries originated from the impact of strong selection. Here, we assessed the extent of LD in a cattle population from western Africa that was bred in an extensive farming system. The analyses were performed on 363 individuals in a Bos indicus x Bos taurus population using 42 microsatellite markers on BTA04, BTA07 and BTA13. A high level of expected heterozygosity (0.71), a high mean number of alleles per locus (9.7) and a mild shift in Hardy-Weinberg equilibrium were found. Linkage disequilibrium extended over shorter distances than what has been observed in cattle from developed countries. Effective population size was assessed using two methods; both methods produced large values: 1388 when considering heterozygosity (assuming a mutation rate of 10(-3)) and 2344 when considering LD on whole linkage groups (assuming a constant population size over generations). However, analysing the decay of LD as a function of marker spacing indicated a decreasing trend in effective population size over generations. This decrease could be explained by increasing selective pressure and/or by an admixture process. Finally, LD extended over small distances, which suggested that whole-genome scans will require a large number of markers. However, association studies using such populations will be effective.

Consequences of Ti-, Li-, and Er-ion implantations on the optical properties of single layers of Ta(2)O(5).

Tantalum pentoxide (Ta(2)O(5)) layers made by ion plating are implanted with a high fluence of keV Ti, Li, and Er ions. The resulting refractive-index profiles are given from the analysis of guided-wave propagation conditions. A comparison with spectrophotometric measurements is presented. All the implanted layers present low losses (extinction coefficient of some 10(-6)) after thermal annealing in air. Ti-implanted layers exhibit an increase in refractive index, whereas Li- and Er-implanted layers present a slight decrease in refractive index. Er-implanted layers present photoluminescent properties.