Comparative analysis of the Leishmania infantum-specific antibody repertoires and the autoantibody repertoires between asymptomatic and symptomatic dogs

International audienceLeishmania (L.) infantum-infected dogs may present with a large range of clinical signs, from apparently healthy with no or few (asymptomatic dogs, AD) to several clinical signs indicators of active infection (symptomatic dogs, SD). The present study is justified by the conflicting reports describing that either L. infantum-specific IgG1 or IgG2 antibodies may be used as isotype marker of the asymptomatic infection status and by the lack, to our knowledge, of previous analysis of the IgG sub-classes autoantibody repertoires of Leishmania-infected dogs. On the basis of clinical evaluation and laboratory testing (IFAT, parasitological examination of Giemsa-stained lymph node smears, L. infantum antigens-ELISA of total (Tot) IgG), 131 dogs were categorized as SD, asymptomatic seronegative (AND) or seropositive dogs (APD) from surrounding areas, and as negative control dogs (CTD). ELISA based on leishmanial native antigens or recombinant LACK and LeIF proteins showed that SD produce higher levels of specific Tot IgG, IgG1 and IgG2 antibodies than APD, and that for both clinical stages, the antibody titers of IgG2 isotype were constantly higher than those of the IgG1. The seroprevalences of Tot IgG, IgG2 did not differ between APD and SD groups (97 and 97% in SD; 100 and 96% in APD, respectively) whereas that of IgG1 was slightly lower in SD (88% of APD versus 82% of SD). The autoantibody repertoires were analyzed by ELISA using HEp-2 extracts, ds-DNA, human albumin and transferrin as self-antigens and by Western blot using HEp-2 proteins. ELISA results' indicated that APD develop higher levels of IgG1 autoantibodies, and higher seroprevalence (50% and 26% in APD and SD, respectively), contrasting with lower levels and seroprevalences of Tot IgG and IgG2 (43 and 68% for APD; 100 and 74% for SD). Interestingly, SD showed a stronger IgG1 and particularly IgG2 reactivity with transferrin, an iron-binding protein, than APD and AND. Western blotting experiments produced heterogeneous IgG1 and IgG2 inter- and intra-groups reactivity profiles towards HEp-2 proteins, to identify a specific antigenic profile. Generated data from competitive HEp-2-ELISA using leishmanial antigens as inhibitors were in favor that IgG1 antibodies are predominantly autoantibodies to self-antigens in APD whereas they are mainly cross-reactive (Leishmania/self-antigens) in SD

Cultivation, cryopreservation and resuscitation of Theileria annulata transformed cells in serum-free media

Introduction: Tropical theileriosis is a protozoan disease caused by Theileria annulata that affects cattle in Northern Africa, the Middle East and Asia where vector ticks of the genus Hyalomma occur. Various measures are applied to control the disease, including vaccination with attenuated T. annulata schizonts. Cultivation of T. annulata schizonts is mainly conducted in media containing Fetal Bovine Serum (FBS), which has some disadvantages such as costs, batch- to-batch variation and ethical concerns. Methods: In this study, we conducted three experiments to evaluate the ability of (1) T. annulata strains grown in RPMI with 10% FBS (RPMI-FBS) to adapt and grow in serum-free media (i.e., HL-1, RPMI without FBS supplementation, ISF-1, and M199), (2) a T. annulata strain grown in ISF-1 and subsequently frozen in this medium to grow in ISF-1 again after long-term storage in liquid nitrogen, and (3) a T. annulata strain freshly isolated from infected bovine lymphocytes to growin ISF-1, also after cryopreservation. Cell numbers, schizont index, the viability and generation doubling time were calculated in all experiments. Results and discussion: In the first experiment, the Hessiene and Beja cell lines from Tunisia previously cultivated in RPMI-FBS and adapted to serum-free media continued to grow significantly better in RPMI-FBS compared to the serum-freemedia. In the second experiment, a Tunisian cell line (Hessiene) cryopreserved in ISF-1 with 5%[v/v] dimethylsulfoxide (DMSO) grewbetter after thawing in RPMI-FBS compared to ISF-1 with a highly significant difference in cell growth (p < 0.001), whereas the third experiment showed that the Ankara cell line had similar growth characteristics in both RPMI-FBS and ISF-1 before and after thawing, with a shorter generation doubling time in ISF-1 than in RPMI-FBS (p = 0.23). Our findings suggest that freshly isolated cells can be propagated, frozen and thawed in serum-free media such as ISF-1, but once cells are adapted to cultivation in the presence of FBS or resuscitated from frozen storage, propagation in serum-free media may not perform as well as cultivation in RPMI-FBS

Dual RNA-seq to catalogue host and parasite gene expression changes associated with virulence of T. annulata-transformed bovine leukocytes: towards identification of attenuation biomarkers

The apicomplexan parasite Theileria annulata is transmitted by Hyalomma ticks and causes an acute lymphoproliferative disease that is invariably lethal in exotic cattle breeds. The unique ability of the schizont stage of T. annulata to transform infected leukocytes to a cancer-like phenotype and the simplicity of culturing and passaging T. annulata-transformed cells in vitro have been explored for live vaccine development by attenuating the transformed cells using lengthy serial propagation in vitro. The empirical in vivo evaluation of attenuation required for each batch of long-term cultured cells is a major constraint since it is resource intensive and raises ethical issues regarding animal welfare. As yet, the molecular mechanisms underlying attenuation are not well understood. Characteristic changes in gene expression brought about by attenuation are likely to aid in the identification of novel biomarkers for attenuation. We set out to undertake a comparative transcriptome analysis of attenuated (passage 296) and virulent (passage 26) bovine leukocytes infected with a Tunisian strain of T. annulata termed Beja. RNA-seq was used to analyse gene expression profiles and the relative expression levels of selected genes were verified by real-time quantitative PCR (RT-qPCR) analysis. Among the 3538 T. annulata genes analysed, 214 were significantly differentially expressed, of which 149 genes were up-regulated and 65 down-regulated. Functional annotation of differentially expressed T. annulata genes revealed four broad categories of metabolic pathways: carbon metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum and biosynthesis of secondary metabolites. It is interesting to note that of the top 40 genes that showed altered expression, 13 were predicted to contain a signal peptide and/or at least one transmembrane domain, suggesting possible involvement in host-parasite interaction. Of the 16,514 bovine transcripts, 284 and 277 showed up-regulated and down-regulated expression, respectively. These were assigned to functional categories relevant to cell surface, tissue morphogenesis and regulation of cell adhesion, regulation of leucocyte, lymphocyte and cell activation. The genetic alterations acquired during attenuation that we have catalogued herein, as well as the accompanying in silico functional characterization, do not only improve understanding of the attenuation process, but can also be exploited by studies aimed at identifying attenuation biomarkers across different cell lines focusing on some host and parasite genes that have been highlighted in this study, such as bovine genes (CD69, ZNF618, LPAR3, and APOL3) and parasite genes such as TA03875

Sequence polymorphisms in a Theileria annulata surface protein (TaSP) known to augment the immunity induced by live attenuated cell line vaccine

Theileria annulata is a tick‐borne protozoan causing tropical theileriosis in cattle. The use of attenuated cell line vaccines in combination with subunit vaccines has been relatively successful as a control method, as exemplified by a recent study in which immunization with a local cell line followed by booster vaccinations with recombinant T. annulata surface protein (TaSP) resulted in 100% protection upon field challenge in Sudan. However, these findings cannot be directly extrapolated to other countries as culture‐attenuated live vaccines are generated using local strains and no systematic evaluation of genotype differences between countries has been undertaken. In this study, we sequenced the TaSP gene from T. annulata cell lines and field isolates from Tunisia (n = 28) and compared them to genotypes from Sudan (n = 25) and Morocco (n = 1; AJ316259.1). Our analyses revealed 20 unique TaSP genotypes in the Tunisian samples, which were all novel but similar to genotypes found in Asia. The impact of these polymorphisms on the ability of the TaSP antigen to boost the immunity engendered by live cell line vaccines, especially in Tunisia where studies with TaSP have not been conducted, remains to be examined. Interestingly, phylogenetic analyses of publicly available TaSP sequences resolved the sequences into two clusters with no correlation to the geographical origin of the isolates. The availability of candidate vaccines that were recently attenuated using local strains from Sudan, Tunisia, Egypt and Morocco should be exploited to generate a comprehensive catalogue of genetic variation across this regional collection of attenuated live vaccines

Genotype-specific Patterns of Physiological, Photosynthetic, and Biochemical Responses in Faba Bean Contrasting Pair to Salinity

To understand the salinity tolerance mechanism in faba bean contrasting pair to salinity (cv. Chourouk as sensitive and cv. Najeh as tolerant), we evaluated the effect of high salt concentration (150 mM NaCl) on the photosynthetic, physiological, and biochemical parameters at short and long term of treatment (1 and 6 days, respectively) in the seedling stage. In general, the salinity affects the growth of plants. High salinity decreased all studied parameters, especially transpiration rate (E), stomatol conductance (gs), net CO2 assimilation (A), and substomatal CO2 concentration (Ci), and dramatic changes was registered in cv. Chourouk compared to cv. Najeh. Chlorophyll contents were also affected by salinity, especially in the sensitive variety. In addition, the synthesis of osmolytes (proline) was determinate, to understand whether the osmotic adjustment is a mechanism used by cv. Najeh to tolerate salt stress. Our research suggests that cv. Najeh should be introduced in a crossbreeding program as an elite salt-tolerant germplasm

In vitro feeding of all life stages of two-host Hyalomma excavatum and Hyalomma scupense and three-host Hyalomma dromedarii ticks

Ticks are blood-sucking ectoparasites and can transmit various pathogens of medical and veterinary relevance. The life cycle of ticks can be completed under laboratory conditions on experimental animals, but the artificial feeding of ticks has attracted increased interest as an alternative method. This study represents the first report on the successful in vitro feeding of all life stages of two-host tick species, Hyalomma scupense and Hyalomma excavatum, and the three-host tick Hyalomma dromedarii. The attachment and engorgement rates of adults were 84% (21/25) and 76% (19/25) for H. scupense females. For adult H. excavatum and H. dromedarii, 70% (21/30) and 34.4% (11/32) of the females attached and all attached females successfully fed to repletion. The oviposition rates of the artificially fed females were 36.4%, 57.1% and 63.1% for H. dromedarii, H. excavatum and H. scupense, respectively, with a reproductive efficiency index varying between 44.3 and 60.7%. For the larvae, the attachment and engorgement rates were 44.2% (313/708) and 42.8% (303/708) for H. dromedarii, 70.5% (129/183) and 56.8% (104/183) for H. excavatum and 92.6% (113/122) and 55.7% (68/122) for H. scupense. The attachment and engorgement rates for the nymphs were 90.2% (129/143) and 47.6% (68/143) for H. dromedarii, 66.7% (34/51) and 41.2% (21/51) for H. excavatum, and 44.1% (30/68) and 36.8% (25/68) for H. scupense. Molting rates of the immature stages varied between 71.3% (216/303) and 100% (68/68) for the larvae and between 61.9% (13/21) and 96% (24/25) for the nymphs. The successful in vitro feeding of all stages of the three Hyalomma species makes this method a valuable tool for tick research, with potential applications in studies on the pathogens transmitted by these tick species such as Theileria annulata

Detection and molecular identification of Anaplasma phagocytophilum and Babesia spp. infections in Hyalomma aegyptium ticks in Tunisia

Tortoises of the genus Testudo are the main hosts of Hyalomma aegyptium ticks. This species serves as a vector of several zoonotic pathogens. Therefore, the present study aimed to investigate the presence of four pathogens associated with H. aegyptium ticks obtained from tortoises from Tunisia. Conventional, multiplex and nested PCRs were used for Aanaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii and Babesia spp. screening. The molecular analyses revealed the presence of A. phagocytophilum and Babesia spp. None of the ticks, were infected by E. canis or C. burnetii species. Co-infection was detected in four ticks. As a conclusion, this is the first detection of A. phagocytophilum and Babesia spp. in H. aegyptium ticks collected from tortoises, in Tunisia. Thus, considering these results, the spur-thighed tortoise constitute a potential host of H. aegyptium which plays an important role in the transmission of pathogenic agents affecting both human and animals. In term of public health, a strict control and surveillance should be carried to reduce the circulation of such pathogens between different hosts

Development and Evaluation of a Loop-mediated Isothermal Amplification Assay for Rapid Detection of Theileria annulata Targeting the Cytochrome B Gene.

International audienceBackground : Theileria annulata is an economically important cattle disease in North Africa that occurs in subtropical and tropical areas. Accurate and rapid, molecular diagnosis of tropical theileriosis is an important issue that allows early treatment and, prevents transmission. We developed and validated a Theileria annulata specific LAMP assay targeting the cytochrome b multicopy gene, in order to increase the DNA detection sensitivity.Methods : The methodology was used to evaluate the occurrences of T. annulata in 88 field samples collected in Northern Tunisia during 2013-2014. The specificity and sensitivity of the LAMP assays were compared to conventional cytochrome b PCR and routine microscopy commonly used on naturally infected cattle blood samples.Results : The PCR assay showed a sensitivity of 70% and specificity around 75%. Our LAMP assay showed a suitable sensitivity 78.7% and specificity 87.5%, with, however, positive (98.4%) and negative (29.1%) predictive values.Conclusion : The LAMP assay is a simple and convenient diagnostic tool for tropical theileriosis. Moreover, LAMP does not require experienced staff and specialized equipment for sampling procedures and it is practical outside laboratories and can be used for field diagnosis

Monitoring the Aroma Compounds of <em>Vicia faba</em> L var. Major and var. Minor

The volatile compounds of Vicia faba. L var. minor and var. major seeds were evaluated by headspace-solid phase micro-extraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). The total identification percentages of the extracted volatiles were 95.5% and 98.3%, respectively. The number of aroma compounds detected was 28. Among them, 15 compounds were determined in the emission of whole legume seeds of minor cultivar and 22 from the major one. The volatiles were classified into five chemical classes, i.e., monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons, apocarotenes, and non-terpene derivatives. Aldehydes and alkanes were considered as the most abundant constituents in non-terpene derivatives, followed by esters, alcohols, phenols, phenones, and hydrocarbons. A wide difference in term of volatiles was observed between major and minor faba bean cultivars. This study can provide useful information about the specific volatile characteristics for each cultivar and its possible use in the conception of legume-based ingredients and for pertinent breeding programs

In vivo evidence for the resistance of Theileria annulata to buparvaquone.

International audienceThe present study describes an outbreak of tropical theileriosis cases refractory to buparvaquone treatment, which occurred in a small-size dairy farm in Tunisia. Out of seven treated cows, four died in spite of repeated buparvaquone injections (2.5 and 5 mg kg(-1)) and the monitoring of the affected cows showed no improvement of the course of the disease with a consistent decrease in the haematocrit, the persistence of fever and an increased parasitaemia after treatment. Ticks were fed on a calf experimentally infected with one isolate established in culture from one of the cases and the resultant infected ticks ground up to generate a supernatant infected with the potentially resistant stock. This was used to experimentally infect three calves, and the clinical observations, post-buparvaquone treatment, showed an absence of the usual effect of buparvaquone treatment on the parasite Theileria annulata, such as the rapid decline of schizont index and parasitaemia and a rapid recovery from the disease. These results confirmed for the first time the occurrence of resistance to buparvaquone in the protozoan T. annulata