TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

Activation constitutive de JNK et AP-1 dans les lymphocytes parasités par Theileria (leur rôle dans la survie et la transformation cellulaire)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Use of micro-rotation imaging to study JNK-mediated cell survival in Theileria parva-infected B-lymphocytes

Lymphocytes infected with the protozoan parasite Theileria parva are transformed to permanently proliferating cells, an event underlying the pathology of the disease. However, the molecular signalling mediating this process is complex and poorly understood. Here, we show that down-regulation of JNK signalling by transient over expression of a dominant-negative mutant of JNK (JNK-APF) significantly increases Annexin-V-phycoerythrin (V-PE) labelling on infected B cell populations observed using flow cytometry. To establish whether this increase was specifically due to apoptosis, we used a novel single-cell imaging method: micro-rotation (MR)-imaging, designed to allow high-resolution 3-dimensional imaging of single cells in suspension. With this method we visualized subcellular patterns of V-PE uptake and chromatin organization in lymphocytes co-transfected with JNK-APF and GFP-tagged histone-H2B. This single-cell approach allowed us to clearly reveal characteristic apoptotic phenotypes, whose patterns reflected progressive states of programmed cell death due to JNK down-regulation. Our results strongly suggest a role for JNK in the survival of Theileria-infected B cells, and demonstrate the powerful utility of a new and unique 3-dimensional imaging method for living cells in suspension.Peer Reviewe

Laboratory evaluations of the immediate and sustained effectiveness of lotilaner (Credelio™) against three common species of ticks affecting dogs in Europe

Abstract Background There is a continuing need for novel approaches to tick control in dogs. One such approach lies in the ability of lotilaner (Credelio™), an isoxazoline with a rapid onset of action, to provide sustained efficacy against ticks. Two studies were undertaken to confirm lotilaner’s efficacy, at the minimum dose rate of 20 mg/kg, against the three most common tick species in Europe. Methods In each of two studies, 16 Beagle dogs, at least 6 months old, were ranked and blocked by tick counts from infestations placed approximately 1 week before treatment. Within blocks, dogs were randomized to receive either lotilaner flavoured chewable tablets at as close as possible to, but not less than the minimum dose rate of 20 mg/kg, or to be sham-treated controls. Study 1 assessed lotilaner efficacy against concurrent infestations with 50 (± 6) Rhipicephalus sanguineus and 70 (± 6) Ixodes ricinus; Study 2 infestations were with 50 (± 2) Dermacentor reticulatus. Infestations were performed on Day -2 with counts on Day 2, 48 (± 2) hours post-treatment. Post-treatment infestations were performed on Days 7, 14, 21, 28 and 35, and ticks were counted 48 (±2) hours post-infestations. Efficacy was determined by the percent reduction in mean live tick counts. Results Control group infestations for each tick species were adequate for assessing lotilaner efficacy at all assessment times. On Day 2 no live ticks were found on any lotilaner-treated dog. For subsequent counts, in Study 1 lotilaner was 100% effective in eliminating live I. ricinus and R. sanguineus on all but two occasions for each tick; on each of those occasions efficacy was sustained at greater than 98.0%. In Study 2, except for a single unattached live tick found on Day 16, efficacy against D. reticulatus was 100% at every post-treatment assessment. Conclusion The high and sustained efficacy against the three common species of ticks in Europe, R. sanguineus, I. ricinus and D. reticulatus, demonstrates that lotilaner can be a valuable tool in the treatment of canine tick infestations. Lotilaner flavoured chewable tablets were well tolerated and effectiveness was sustained through at least 35 days

Molecular and serological rapid tests as markers of Trypanosoma cruzi infection in dogs in Costa Rica

Chagas disease is a zoonotic disease caused by Trypanosoma cruzi and dogs are one of the main domestic reservoirs. Materials and Methods: One molecular (OligoC‑TesT, Coris Bioconcept) and one serological (T. cruzi‑Detect, Inbios) rapid tests were evaluated as infection markers for T. cruzi in 102 dogs living in eight villages endemic for Chagas in Costa Rica. Results: T. cruzi‑Detect performed well as screening tool with 23.3% positive samples. The large number of invalid results (66.7%) observed in samples tested with OligoC‑TesT precluded assessing the use of this new method as epidemiological tool to detect T. cruzi infection in dogs.La enfermedad de Chagas es una enfermedad zoonótica causada por el Trypanosoma cruzi y los perros son uno de los principales reservorios domésticos. Materiales y métodos: Un molecular (OligoC-TesT, Coris Bioconcept) y una serológica (T. cruzi-Detect, Inbios) de T. cruzi en 102 perros que viven en ocho pueblos endémicos de Costa Rica. de Chagas en Costa Rica. Resultados: T. cruzi-Detect funcionó bien como herramienta de cribado con un 23,3% de muestras positivas. El gran número de resultados no válidos (66,7%) observado en las muestras analizadas con OligoC-TesT impidió evaluar el uso de este nuevo método como herramienta epidemiológica para detectar la infección por T. cruzi en perros.Universidad Nacional, Costa RicaEscuela de Medicina Veterinari

MHC-specific cytotoxicity of stimulated PBMC.

<p><b>All sheep samples were assayed similarly and two representative data are shown.</b> (a) Sheep 4223 effectors lysed autologous infected cells more effectively than class I MHC-mismatched infected cells from Sheep 4247 at all effector: target ratios. (b) Sheep 4247 effectors lysed autologous infected cells, but not class I MHC mismatched infected cells from Sheep 4223.</p

CD8⁺ T cell response to 13 <i>T</i>. <i>lestoquardi</i> antigens measured by IFNγ ELISA.

<p>Sheep 4247 fibroblasts were transfected with expression constructs of candidate antigen genes using either Lipofectamine LTX or Fugene transfection reagents for 48 h before the addition of effector cells for 72 h and IFNγ ELISA. The antigens were tested separately and data are presented as mean ± SD of biological repeats (n = 3).</p

MHC class I and II alleles identified for individual sheep.

<p>MHC class I and II alleles identified for individual sheep.</p

Tl8 and Tl9 peptide screen for CTL epitopes.

<p>(a) Peptides positively identified from peptide pools were incubated at 1 μg ml-1 with fibroblasts and assayed for IFNγ production by autologous stimulated effector cells. <i>T</i>. <i>lestoquardi</i>-infected cells incubated with effectors served as a positive control and effectors only served as a negative control. (b) Tl8 peptides were titrated and assayed for IFNγ production by autologous stimulated effector cells, which increased with increasing concentrations of peptides. (c) Overlapping peptide sequence of H04 and E01 of Tl8. (d) Overlapping peptide sequence of E06 and E07 of Tl9.</p