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

Novi sublingviformni vulvarni zalistak oblića Haemonchus sp. u prirodno invadiranih domaćih preživača zaklanih u klaonici Béja u sjevernom Tunisu.

The present study was conducted to determine the type of vulvar morphology in female Haemonchus worms collected from the abomasa of sheep, goats and cattle slaughtered for human consumption in Béja Abattoir in Tunisia with a Mediterranean type of climate. For the purpose, a total of 2450 female Haemonchus worms from abomasa of 363 sheep, 258 goats and 152 cattle were thoroughly examined for the types of their vulvar flap. Of the total 143 female Haemonchus worms from cattle 21.5% linguiform, 41.8% knobbed and 3.4% smooth vulvar flap types were recorded. Out of the total 645 female Haemonchus worms from abomasa of sheep, 42.5% linguiform, 36.3% knobbed and 21.2% smooth vulvar morph types were identified. In goats a total of 1662 female worms were differentiated into 27.7% linguiform, 65.8% knobbed and 6.4% smooth vulvar morph types. Further subclassification of 43 linguiform vulvar flap female Haemonchus worms from cattle revealed an overall proportion of 11.5% LA type, 26.3% LB type, 17.1% LC type, 0.8% LD type and 11.0% LI linguiform subtypes. Likewise, from sheep, of the total of 327 linguiform vulvar flap female worms, 31.6% LA type, 18% LB type, 26.3% LC type, 3.9% LD type and 3.5% LI linguiform subtypes were differentiated. From goats, of the total of 461 linguiform vulvar flap female worms, 48% LA type, 13.8% LB type, 21.6% LC type, 4.4% LD type and 12.3% LI linguiform subtypes were detected. The LB sublinguiform type had the highest overall proportion in cattle, whereas the LA sub linguiform type was the most predominant in both sheep and goats. An interesting finding of the current study is the documentation of a new type of sublinguiform vulvar flap type in female Haemonchus worms for the first time, from the Mediterranean type of climate in Tunisia. To the best of our knowledge this is the first report of new vulvar morphology in female Haemonchus worms, designated as LD sublinguiform vulvar flap with three cuticular inflations. Further detailed studies are urgently needed on the prevalence, morphological, molecular and genetic characterization of Haemonchus spp. with different vulvar flap types in different agro-ecologies, animal species and breeds, and management systems in Tunisia and other countries.Istraživanje je poduzeto kako bi se odredio morfološki tip ženki oblića roda Haemonchus sakupljenih iz sirišta ovaca, koza i goveda zaklanih u klaonici Béja na području Tunisa s mediteranskom klimom. Ukupno je bilo izdvojeno 2450 ženki oblića roda Haemonchus iz sirišta 363 ovce, 258 koza i 152 goveda. Nakon izdvajanja određivane su razlike u izgledu vulvarnog zaliska. Od ukupno 143 ženke izdvojene iz sirišta goveda 21,5% je imalo lingviformni tip zalistaka, 41,8% zalistak s izbočinom dok je samo 3,4% imalo glatke zaliske. Među oblićima (645) izdvojenima iz ovaca ustanovljeno je 42,5% s lingviformnim zaliskom, 36,3% sa zaliskom s izbočinom i 21,2% oblića s glatkim zaliskom. Mikroskopskim pregledom 1662 ženke izdvojene iz koza u njih 27,7% ustanovljeni su lingviformni zalisci, u 65,8% zalisci s izbočinom te u 6,4% glatki zalisci. Daljnjim svrstavanjem 43 ženke s lingviformnim zaliscima podrijetlom iz goveda pokazalo se da 11,5% pripada LA podtipu, 26,3% LB podtipu, 17,1% LC podtipu, 0,8% LD podtipu i 11,0% LI podtipu. Među 325 oblića s lingviformnim zaliscima izdvojenih iz ovaca 31,6 % je bilo LA podtipa, 18% LB podtipa, 26,3% LC podtipa, 3,9% LD podtipa i 3,5% LI podtipa. Među 461 oblićem s lingviformnim zaliscima podrijetlom iz koza 48% bilo je LA tipa, 13,8% LB tipa, 21,6% LC tipa, 4,4% LD tipa i 12,3% LI podtipa. LB tip zaliska bio je najučestaliji u oblića izdvojenih iz goveda dok je LA bio najčešći tip zaliska u oblića izdvojenih iz ovaca i koza. Ovo je prvi nalaz novog tipa sublingviformnih vulvarnih zalistaka u ženki oblića roda Haemonchus na području Tunisa s mediteranskom klimom. Taj zanimljiv novi nalaz sublinguiformnog vulvarnog zaliska s tri kutikularna udubljenja nazvan je LD tip

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

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

Toxoplasma gondii infection and toxoplasmosis in North Africa: a review

Toxoplasmosis is an important zoonosis caused by an obligate intracellular parasitic protozoan, Toxoplasma gondii. The disease is distributed worldwide and can affect all warm-blooded vertebrates, including humans. The present review aimed to collect, compile and summarize the data on the prevalence of T. gondii infection in humans and animals in the five North African countries (Morocco, Algeria, Tunisia, Libya and Egypt). Published data from national and international databases were used. Distribution patterns and risk factors for T. gondii infection are discussed, focusing on biotic and abiotic factors. This review is a comprehensive epidemiological analysis of T. gondii infection in North Africa and will therefore be a useful tool for researchers. It can also be used to propose or enhance appropriate national toxoplasmosis control programs

La theileriose tropicale des bovins (Theileria annulata) en tunisie (caractérisation des états d'endémie et deloppemnt de lignées de schizontes attebues)

La première partie de ce travail est une revue bibliographique sur Theileria annulata et la theilériose tropicale, où les particularités de cette protozoose en Tunisie sont présentées. La deuxième partie, expérimentale, est divisée en deux chapitres. Le premier chapitre présente les résultats d'enquête séro-épidémiologiques sur la theilériose dans les élevages des zones bioclimatiques sud-humide et semi-aride. Trois états d'endémie ont été identifiés en Tunisie : un état d'endémie stable dans moins de 5% des étables étudiées, et deux états d'endémie instable : modérée et élevée. L'endémie instable modérée existe dans près de 20% des étables et se caractérise par des risques potentiels maximaux de theilériose tropicale chez les bovins en 2ème et 3ème saison estivale. L'instabilité élevée est rencontrée dans 75% environ des élevages, associée à des risques maximaux de maladie chez les vaches laitières de 4 saisons estivales et plus. Les implications de ces données dans le choix de souches atténuées vaccinales et l'élaboration d'une stratégie de vaccination de masse sont discutées. Le deuxième chapitre décrit l'atténuation de trois lignées parasitaires autochtones de T. annulata en culture cellulaire à long terme de leucocytes infectés par des schizontes. L'atténuation de trois lignées parasitaires autochtones de T. annulata en culture cellulaire à long terme de leucocytes infectés par des schizontes. L'atténuation est associée à une restriction du polymorphisme parasitaire. L'immunité conférée par ces lignées atténuées envers des infections d'épreuve hétérologues létale et sub-létale a été évalué à 30 jours et à six mois de la vaccination, elle s'est avérée moins solide qu'avec les lignées parentales non atténuées. Le phénotypage des leucocytes infectés par des schizontes en culture e révélé qu'il s'agissait principalement de cellules macrophagiques monocytaires et secondairement de lymphocytes B. L'atténuation est associée à une hyper-expression du marqueur CD25 (récepteur alpha de l'interleukine 2) régulée par le parasite et maintenue après réplication sexué chez la tique, et à une expression réduite du marqueur bovin CD9 like. La transmission aux tiques vecteurs a été testée en conditions expérimentales pour deux des trois lignées atténuées. L'une d'entre elles a été transmise de façon répétée à des tiques Hyalomma anatolicum excavatum associée à un phénomène de ré-expression de virulence. ces différents résultats sont discutés en insistant sur leurs implications pour le développement et l'utilisation d'un vaccin vivant atténué contre la theilériose tropicale en Tunisie.TOULOUSE-ENSAT-Documentation (315552324) / SudocSudocFranceF

Prevalence of

Dermanyssus gallinae (Mesostigmata: Dermanyssidae), a mite of poultry, represents the most important ecotoparasite of egg-laying poultry in several countries. We estimated the prevalence of D. gallinae infestation in 38 industrial poultry farms (28 egg-laying and 10 reproductive hen farms) in the governorate of Nabeul (North-East Tunisia). Traps were placed in two locations of each farm during 24 h in August. The overall prevalence at the farms was estimated to be 34%. A total number of 329 D. gallinae were collected, giving an intensity of 0.0028 and an abundance of 0.0015. Infestation intensity and abundance were significantly higher in egg production farms than reproductive farms. There was no correlation between the intensity of infestation and temperature. An exponential correlation was observed between the birds’ age and infestation intensity. We recommend a systematic survey of poultry farms during the whole breeding period. Prompt treatment is recommended to avoid the exponential increase of mite population

PARASITES BOVINS ALGERIE CEVA 2018.pptx

Présentation sur les parasites des bovins en Algérie