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.

[Non-fatal disseminated mucormycosis in a solid organ transplant]. / Mucormycose disséminée d'évolution favorable chez une greffée pulmonaire.

BACKGROUND: Mucormycosis is a rare fungal infection occurring most frequently in immunocompromised patients. The pathogens are filamentous fungi, order of Mucorales. Disseminated mucormycosis is a severe, life treating disease. Early diagnosis is a major determinant for prognosis, however, it remains difficult. The management consists in an early antifungal therapy using lipid formulation of amphotericin B associated with an extensive surgical debridement. Despite this therapeutic of choice, the mortality of disseminated mucormycosis remains high. OBSERVATION: We report the case of disseminated mucormycosis in a 25 years old woman 9 months after a pulmonary transplantation. The clinical presentation included pulmonary and thyroid localization and the pathogen was Absidia corymbifera. The patient survived thanks to a large surgical debridement, and an early antifungal bitherapy by lipid formulation of amphotericin B and posaconazole. CONCLUSION: The re-emergence and the high mortality of mucormycosis in solid organ transplant receiver show the necessity to find new therapeutic approaches. Posaconazole associated with liposomal amphotericin B could be an interesting option to treat disseminated mucormycosis and improve their outcome.

Sequence analysis of the large (L) polymerase gene and trailer of the peste des petits ruminants virus vaccine strain Nigeria 75/1: expression and use of the L protein in reverse genetics.

The large (L) polymerase gene and the 5'-terminal UTR of the genome of peste des petits ruminants virus (PPRV), vaccine strain Nigeria 75/1, were cloned and sequenced. The L protein was also expressed in eukaryotic cells and its polymerase activity was quantitatively measured in a PPR reverse genetics assay using a reporter minigenome. Comparative sequence analysis of this functional L gene with corresponding genes of other morbilliviruses showed a degree of conservation exceeding 70%. The multiple sequence alignment and the phylogenetic study of L gene discriminated the morbilliviruses in 6 clusters, which are more closely related to Tupaia and Henipaviruses than to other paramyxoviruses. Important protein domains and functional motifs of the L polymerase of the PPRV Nigeria 75/1 vaccine were also identified by using different bioinformatics tools.

[Morbillivirus infections of ruminants: the nearly eradicated rinderpest and the "peste des petits ruminants", an expanding disease in the South and a threat for Europe]. / Infections à Morbillivirus chez les ruminants: la peste bovine en voie d'éradication et la peste des petits ruminants en extension vers le Nord.

Rinderpest (RP) and peste des petits ruminants (PPR) are contagious viral diseases of domestic and wild ruminants producing high mortality. They are caused by viruses belonging to the Morbillivirus genus, Paramyxoviridae family. Control tools (vaccines and specific diagnostic tests) exist for these two diseases. They have been successfully used during the global rinderpest eradication programme (GREP) and the disease is expected to be eradicated by 2010. In contrast, a similar programme does not exist for PPR, which is still spreading in Africa and Asia. The persistence of PPR in Turkey and its recent introduction in Morocco, make the disease a real threat for Europe. Improvement of control measures against PPR would benefit from the development of a marker vaccine and its companion serological test, thus allowing the differentiation between infected and vaccinated animals (DIVA vaccines and tests). The recent development of reverse genetics for morbilliviruses offers this new possibility.

The threat of peste des petits ruminants: progress in vaccine development for disease control.

Peste des petits ruminants (PPR) is a highly contagious animal disease caused by a virus in the genus Morbillivirus, family Paramyxoviridae. This infection is responsible for high morbidity and mortality in sheep and goats and in some small wild ruminant species. The huge number of small ruminants, which are reared in the endemic areas makes PPR a serious disease threatening the livelihood of poor farmers. Taking advantage of the closely relationship between rinderpest and PPR viruses, the attenuated rinderpest vaccine was used in the control of PPR. It is now replaced by the homologous attenuated PPR vaccine. Unfortunately, animals that have received this vaccine cannot be distinguished serologically from infected animals. With the advent of DNA recombinant technology, efforts are being made to develop effective PPR marker vaccines to enable such differentiation and which would allow countries to implement both vaccination and disease surveillance programmes at the same time.

Peste des petits ruminants (PPR) outbreak in Tajikistan.

The occurrence of outbreaks of peste des petits ruminants (PPR) in three districts of Tajikistan is described. The causal strain (PPR Tajikistan) was characterized and the sequence of its N gene was compared with that of 43 other strains isolated since 1968 in Africa, the Middle East and Asia. The study demonstrated (1) the value of the N gene as a target in comparing isolates obtained over an extended period of evolution, and (2) that clustering was related to the geographical origin of strains.

Development of a dual recombinant vaccine to protect small ruminants against peste-des-petits-ruminants virus and capripoxvirus infections.

A recombinant capripoxvirus vaccine containing a cDNA of the peste-des-petits-ruminants virus (PPRV) fusion protein gene was constructed. A quick and efficient method was used to select a highly purified recombinant virus clone. A trial showed that a dose of this recombinant as low as 0.1 PFU protected goats against challenge with a virulent PPRV strain.

Goat immune response to capripox vaccine expressing the hemagglutinin protein of peste des petits ruminants.

Sheep-pox and capripox are contagious diseases of domestic small ruminants for which the causal agent is a poxvirus classified into the Capripoxvirus genus. Viruses of this group have a host range specific to sheep, goats, cattle, and possibly buffalo. Thus, they are clearly indicated as vectors for the development of recombinant vaccines for peste des petits ruminants (PPR). Here we report the immune response of goats inoculated with a recombinant capripox-PPR hemagglutinin.

Functional expression of the catalytic domains of two cysteine proteinases from Trypanosoma congolense.

The catalytic domains of two closely related cysteine proteinases (CP1 and CP2) from Trypanosoma congolense, referred to as C1 and C2, were expressed as proforms in Escherichia coli (C1) and in the baculovirus system (C1 and C2). While the bacterial expression system did not allow recovery of active C1, the baculovirus system led to secretion of inactive zymogens which could be processed at acidic pH into mature enzymes. Active C1 and C2 were purified from serum-free culture supernatants by anion-exchange chromatography and characterised. Their kinetic parameters and pH activity profiles confirmed the relatedness between C2 and native CP2 (congopain). These properties also underline major functional differences between C1 and C2, that appear to relate to discrete but essential sequence differences. It is likely that these two enzymes perform distinct roles in vivo, in the parasite and/or in the host-parasite relationships.