In vitro and in vivo anthelmintic effect of essential oil obtained from Thymus capitatus flowers against Haemonchus contortus and Heligmosomoides polygyrus.

Sheep haemonchosis is a disease that causes serious losses in livestock production, particularly with the increase of cases of anthelmintic resistance around the world. This justifies the urgent need of alternative solutions. The aim of this study was to determine the chemical profile, in vitro, and, in vivo, anthelmintic properties of Thymus capitatus essential oil. To evaluate the, in vitro, anthelmintic activity of the T. capitatus EO on Haemonchus contortus, two tests were used: egg hatch assay (EHA) and adult worm motility (AWM) assay. The nematicidal effect of this oil was evaluated, in vivo, in mice infected artificially with Heligmosomoides polygyrus using faecal egg count reduction (FECR) and total worm count reduction (TWCR). Chromatographic characterization of T.capitatus composition using gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of carvacrol (81.16%), as the major constituents. The IC50 values obtained was 1.9 mg/mL in the EHT. In the AWM assay; T. capitatus essential oil achieved 70.8% inhibition at 1 mg/mL after 8 h incubation. The in vivo, evaluation on H. polygyrus revealed a significant nematicidal effect 7 days post-treatment by inducing 49.5% FECR and 64.5% TWCR, using the highest dose (1600 mg/kg). The results of present study, demonstrate that T.capitatus EO possess a significant anthelmintic properties. Furthermore, it could be an alternative source of anthelmintic agents against gastrointestinal infections caused by H. contortus.

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.

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.

Genetic diversity of Hyalomma marginatum in Tunisia is not influenced by the bio-climate.

Ticks are important ectoparasites responsible for the transmission of several pathogens with significant medical, veterinary, and economic impacts. Climate and social changes have generated substantial changes in ticks' distribution, abundance, and activity patterns, including ticks belonging to the Hyalomma marginatum species. Knowledge on the genetic structure and dynamics of H. marginatum populations might contribute to a better understanding of their current and future evolution under the effects of anthropogenic factors and eco-climatic changes. In the present study, we investigated the genetic structure and phylogenetic distribution of H. marginatum across three bioclimatic regions in Tunisia using two mitochondrial markers (16S and 12S rRNA). The molecular investigations were based on 47 adult H. marginatum ticks collected from humid, upper semi-arid, and sub-humid regions of Tunisia. Our results revealed a genetic diversity of 0.278% and 0.809% using the 16S and 12S markers, respectively. The low genetic diversity that we observed raises the hypothesis of a bottleneck event occasioned by a reduction in the size of the tick population under the effects of environmental factors and/or human activities. This hypothesis is supported by the population's demographic history analysis, which revealed a clear deviation from neutrality and supports the occurrence of a bottleneck event followed by a demographic expansion. The fact that most 16S and 12S variability was present in the ticks from the humid bioclimatic zone may suggest that those ticks represent the ancestral population. Overall, the analysis has shown that the phylogenetic clusters do not correspond to the bioclimatic zones.

Development and field evaluation of PCR assays based on minimum length Bm86 cDNA fragments required for Rhipicephalus and Hyalomma tick species delineation.

Introduction: Hyalomma and Rhipicephalus ticks are important genera that can transmit diseases to both animals and humans, including Crimean-Congo hemorrhagic fever, tick-borne encephalitis, and several types of spotted fever. The accurate identification of tick species is essential for the effective control and prevention of tick-borne diseases. However, traditional identification methods based on morphology can be challenging and subjective, leading to errors. The development of DNA markers has provided more precise and efficient methods for tick species identification, but the currently available markers have limitations in their discriminatory power and sensitivity. To address this need for more sensitive and specific markers, this study aimed to identify two minimum sequence fragments required for tick Hyalomma and Rhipicephalus species identification using the Bm86 cDNA marker, which has previously been shown to be in perfect agreement with the current taxonomy of hard ticks based on its complete sequence. Methods: Based on our in silico determination that a minimum sequence of 398 bp for Rhipicephalus spp. (from 1487 to 1884) and 559 bp for Hyalomma species (from 539 to 1097) was necessary for species delineation, two distinct PCR assays were developed to apply these sequences in practice. Results and discussion: Discrimination between species within each genus was achieved through sequence homology and phylogenetic analysis following the sequencing of the two PCR products. Subsequently, their performance was evaluated by testing them on the field-collected ticks of the Hyalomma and Rhipicephalus genera obtained from various host animals in different geographic regions of Tunisia. The use of shorter partial sequences specific to the tick genera Rhipicephalus and Hyalomma, which target the tick's RNA banks, could represent a significant advance in the field of tick species identification, providing a sensitive and discriminatory tool for interspecific and intraspecific diversity analysis.

Cattle co-infection patterns by hemopathogens and their phylogenetic analysis during the tick season in Constantine and Mila, Northeast Algeria.

Tropical theileriosis, babesiosis, and anaplasmosis are the most dominant tick-borne infections in North Africa where they cause significant economic losses in ruminants' industry. The aim of the present work was to study infections and co-infection patterns in 66 cattle with clinical signs of piroplasmosis and/or anaplasmosis in two localities, Beni Hamidene and Grarem Gouga, districts of Constantine and Mila (Northeast of Algeria), respectively. This study was conducted between early May and late September during four years 2017, 2018, 2020, and 2021. PCR showed that the most frequent pathogen in cattle with clinical signs of piroplasmosis and/or anaplasmosis was Theileria annulata (66/66; 100%) followed by Babesia bovis (21/66; 31.8%), Anaplasma marginale (15/66; 22.7%), and Babesia bigemina (3/66; 4.5%) (p < 0.001). Giemsa-stained blood smears examinations revealed that 66.7% (44/66); 10.6% (7/66); and 9.1% (6/66) of cattle were infected by T. annulata, Babesia spp., and A. marginale, respectively (p < 0.001). PCR revealed seven co-infection patterns: T. annulata/A. marginale (15/66; 22.7%), T. annulata/B. bovis (21/66; 31.8%), T. annulata/B. bigemina (3/66; 4.5%), T. annulata/A. marginale/B. bovis (7/66; 10.6%), T. annulata/B. bovis/B. bigemina (2/66; 3%), T. annulata/A. marginale/B. bigemina (1/66; 1.5%), and T. annulata/A. marginale/B. bigemina/B. bovis (1/66; 1.5%). Phylogenetic analyses showed that T. annulata Tams1 and B. bigemina gp45 sequences were identical to isolates from Mauritania and South Africa, respectively. The three A. marginale amplicons obtained herein had 99.63 to 99.88% similarity between them. This study provides data that can be used to improve control programs targeting these cattle hemopathogens.

Diversity and Phylogeny of Cattle Ixodid Ticks and Associated Spotted Fever Group Rickettsia spp. in Tunisia.

Tick-borne rickettsioses are mainly caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the Rickettsia genus. So far, the causative agents of SFG rickettsioses have not been detected in cattle ticks from Tunisia. Therefore, the aim of this study was to investigate the diversity and phylogeny of ticks associated with cattle from northern Tunisia and their associated Rickettsia species. Adult ticks (n = 338) were collected from cattle in northern Tunisia. The obtained ticks were identified as Hyalomma excavatum (n = 129), Rhipicephalus sanguineus sensu lato (n = 111), Hyalomma marginatum (n = 84), Hyalomma scupense (n = 12) and Hyalomma rufipes (n = 2). After DNA extraction from the ticks, 83 PCR products based on the mitochondrial 16S rRNA gene were sequenced and a total of four genotypes for Rh. sanguineus s.l., two for Hy. marginatum and Hy. excavatum and only one for Hy. scupense and Hy. rufipes were recorded, with the occurrence of one, two and three novel genotypes, respectively, for Hy. marginatum, Hy. excavatum and Rh. sanguineus s.l. mitochondrial 16S rRNA partial sequences. The tick DNA was tested for the presence of Rickettsia spp. by using PCR measurements and sequencing targeting three different genes (ompB, ompA and gltA). Of the 338 analyzed ticks, 90 (26.6%), including 38 (34.2%) Rh. sanguineus s.l., 26 (20.1%) Hy. excavatum, 25 (29.8%) Hy. marginatum and one (50%) Hy. rufipes tick, were positive for Rickettsia spp. Based on 104 partial sequences of the three analyzed genes, the BLAST analysis and phylogenetic study showed the infection of Hy. excavatum, Hy. marginatum and Rh. sanguineus s.l. tick specimens with R. massiliae, R. aeschlimannii and R. sibirica subsp. mongolitimonae and one Hy. rufipes tick specimen with R. aeschlimannii. In addition, coinfection with R. massiliae and R. aeschlimannii was reported in one Hy. marginatum and one Rh. sanguineus s.l. tick specimen, while a coinfection with R. massiliae and R. sibirica subsp. mongolitimonae was recorded in one Rh. sanguineus s.l. tick specimen. In conclusion, our study reports, for the first time in Tunisia, the infection of cattle ticks belonging to Hyalomma and Rhipicephalus genera with zoonotic Rickettsia species belonging to the SFG group.

Anaplasma ovis Prevalence Assessment and Cross Validation Using Multiparametric Screening Approach in Sheep from Central Tunisia.

We conducted a 5-month-long screening of Anaplasma spp. and Anaplasma ovis infection in sheep from central Tunisia. During this longitudinal study, we investigated the infection dynamics using both direct and indirect assessments validated with a polymerase chain reaction (PCR) as the gold standard method. The experimental design included 84 male lambs aged from 6 to 8 months, and 32 ewes, both chosen randomly from June to November with a periodicity of 2 weeks approximately between June and September, and 1 month between September and November. A total of 9 field visits were carried out in this period during which animals were clinically examined and biological samples were extracted. Thus, a total of 716 blood smears, 698 sera from the nine sampling dates, as well as 220 blood samples from the first and the ninth sampling dates were collected from apparently healthy lambs and ewes, respectively, and analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and polymerase chain reaction (PCR) assay, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. Sera were analyzed by competitive enzyme-linked immunosorbent assay (cELISA) and PCR, for the detection of Anaplasma antibodies and A. ovis DNA, respectively. The Anaplasma spp. initial seroprevalence rate was 33.3% in lambs and 100% in ewes, and it then flowed in an upward trend to reach a maximum of 52.6% in lambs, whereas in ewes, the Anaplasma spp. seroprevalence rate remained unchanged and equal to 100%. Meanwhile, the A. ovis initial molecular prevalence was 22.6% at the first visit and 26.3% at the last visit in lambs, whereas in ewes, the molecular prevalence rates of A. ovis were higher in both the first and the last visit estimated at 100% and 85.7%, respectively. The Kappa coefficient between cELISA and PCR indicated a moderate level of agreement on the first sampling date (0.67) and a low agreement level on the last (0.43). Furthermore, an exploratory data analysis using a multimodal machine learning approach highlighted the underlying pattern of each analytical technique used in this study. In this prospect, we were able to establish the performance of each technique at detecting Anaplasma spp. in sheep. The combination of these approaches should improve the field assessment while promoting a data-based decision in precision epidemiology. The genetic follow-up test relevant to A. ovis msp4 sequences revealed three different genotypes, two of which were previously described in Italy.

Genetic characterisation of the Theileria annulata cytochrome b locus and its impact on buparvaquone resistance in bovine.

Control of tropical theileriosis, caused by the apicomplexan Theileria annulata, depends on the use of a single drug, buparvaquone, the efficacy of which is compromised by the emergence of resistance. The present study was undertaken to improve understanding of the role of mutations conferring buparvaquone resistance in T. annulata, and the effects of selection pressures on their emergence and spread. First, we investigated genetic characteristics of the cytochrome b locus associated with buparvaquone resistance in 10 susceptible and 7 resistant T. annulata isolates. The 129G (GGC) mutation was found in the Q01 binding pocket and 253S (TCT) and 262S (TCA) mutations were identified within the Q02 binding pocket. Next, we examined field isolates and identified cytochrome b mutations 129G (GGC), 253S (TCT) and 262S (TCA) in 21/75 buffalo-derived and 19/119 cattle-derived T. annulata isolates, providing evidence of positive selection pressure. Both hard and soft selective sweeps were identified, with striking differences between isolates. For example, 19 buffalo-derived and 7 cattle-derived isolates contained 129G (GGC) and 253S (TCT) resistance haplotypes at a high frequency, implying the emergence of resistance by a single mutation. Two buffalo-derived and 12 cattle-derived isolates contained equally high frequencies of 129G (GGC), 253S (TCT), 129G (GGC)/253S (TCT) and 262S (TCA) resistance haplotypes, implying the emergence of resistance by pre-existing or recurrent mutations. Phylogenetic analysis further revealed that 9 and 21 unique haplotypes in buffalo and cattle-derived isolates were present in a single lineage, suggesting a single origin. We propose that animal migration between farms is an important factor in the spread of buparvaquone resistance in endemic regions of Pakistan. The overall outcomes will be useful in understanding how drug resistance emerges and spreads, and this information will help design strategies to optimise the use and lifespan of the single most drug use to control tropical theileriosis.

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.

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.

First insights into the microbiome of Tunisian Hyalomma ticks gained through next-generation sequencing with a special focus on H. scupense.

Ticks are one of the most important vectors of several pathogens affecting humans and animals. In addition to pathogens, ticks carry diverse microbiota of symbiotic and commensal microorganisms. In this study, we have investigated the first Tunisian insight into the microbial composition of the most dominant Hyalomma species infesting Tunisian cattle and explored the relative contribution of tick sex, life stage, and species to the diversity, richness and bacterial species of tick microbiome. In this regard, next generation sequencing for the 16S rRNA (V3-V4 region) of tick bacterial microbiota and metagenomic analysis were established. The analysis of the bacterial diversity reveals that H. marginatum and H. excavatum have greater diversity than H. scupense. Furthermore, microbial diversity and composition vary according to the tick's life stage and sex in the specific case of H. scupense. The endosymbionts Francisella, Midichloria mitochondrii, and Rickettsia were shown to be the most prevalent in Hyalomma spp. Rickettsia, Francisella, Ehrlichia, and Erwinia are the most common zoonotic bacteria found in Hyalomma ticks. Accordingly, Hyalomma ticks could represent potential vectors for these zoonotic bacterial agents.

High Prevalence of Toxoplasma gondii in Industrial Tunisian Poultry Meat.

PURPOSE: Food-borne Toxoplasma gondii infection is ranked among parasitic diseases of global concern. Toxoplasmosis is qualified as a one health disease, since it affects human health, animals, and ecosystems. The present study was performed to estimate the molecular prevalence of T. gondii in industrial poultry meat samples from Tunisia. A total of 140 breast samples were collected from chicken (Gallus gallus domesticus) and turkey (Meleagris gallopavo) and four breeding systems (broiler chickens, reform laying hens, chicken farm label, and broiler turkeys) cover the majority of chicken meat production in Tunisia. METHODS: For each sample, DNA was extracted and T. gondii-specific PCR, targeting the B1 gene was performed. RESULTS: The overall molecular prevalence of T. gondii in poultry meat samples was 20% (28/140; 95% CI: 13.3-26.6%). The highest prevalence was observed in chicken farm label (25.7%; 9/35). Prevalence rates were 20.0% (7/35), 20.0% (7/35), and 14.3% (5/35) in broiler chickens, broiler turkeys, and reform laying hens, respectively (p > 0.05). CONCLUSION: Our finding suggests the involvement of industrial chicken meat, as a potential risk factor of human T. gondii transmission, especially when the latter is consumed undercooked.

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.

Screening and Analysis of Anaplasma marginale Tunisian Isolates Reveal the Diversity of lipA Phylogeographic Marker and the Conservation of OmpA Protein Vaccine Candidate.

Bovine anaplasmosis caused by Anaplasma marginale is a disease responsible for serious animal health problems and great economic losses all over the world. Thereby, the identification of A. marginale isolates from various bioclimatic areas in each country, the phylogeographic analysis of these isolates based on the most informative markers, and the evaluation of the most promising candidate antigens are crucial steps in developing effective vaccines against a wide range of A. marginale strains. In order to contribute to this challenge, a total of 791 bovine samples from various bioclimatic areas of Tunisia were tested for the occurrence of A. marginale DNA through msp4 gene fragment amplification. Phylogeographic analysis was performed by using lipA and sucB gene analyses, and the genetic relationship with previously characterized A. marginale isolates and strains was analyzed by applying similarity comparison and phylogenetic analysis. To evaluate the conservation of OmpA protein vaccine candidate, almost complete ompA nucleotide sequences were also obtained from Tunisian isolates, and various bioinformatics software were used in order to analyze the physicochemical properties and the secondary and tertiary structures of their deduced proteins and to predict their immunodominant epitopes of B and T cells. A. marginale DNA was detected in 19 bovine samples (2.4%). Risk factor analysis shows that cattle derived from subhumid bioclimatic area were more infected than those that originated from other areas. The analysis of lipA phylogeographic marker indicated a higher diversity of Tunisian A. marginale isolates compared with other available worldwide isolates and strains. Molecular, phylogenetic, and immuno-informatics analyses of the vaccine candidate OmpA protein demonstrated that this antigen and its predicted immunodominant epitopes of B and T cells appear to be highly conserved between Tunisian isolates and compared with isolates from other countries, suggesting that the minimal intraspecific modifications will not affect the potential cross-protective capacity of humoral and cell-mediated immune responses against multiple A. marginale worldwide strains.

Infection by haemopathogens and tick infestation of sheep during summer season in Constantine region, Northeast Algeria.

A study on tick infestation of 43 sheep with clinical symptoms of piroplasmosis and anaplasmosis was carried out during summer seasons of 2016 and 2017 in 34 sheep flocks from Beni Hamidene locality, district of Constantine, Northeast Algeria. Only animals with clinical symptoms of piroplasmosis and/or anaplasmosis were checked for tick infestation. Among the 43 examined sheep, 58 ± 15% were infested by ticks. A total of 185 adult ticks (100 males and 85 females) were collected from the 25 sheep. Two tick genera, Rhipicephalus and Hyalomma consisting of four species were collected, Rhipicephalus bursa was the most frequent tick (164/185; 88.6 ± 4.6%), followed by Rhipicephalus sanguineus sensu lato (16/185; 8.6 ± 4%), Rhipicephalus (Boophilus) annulatus (4/185; 2.2 ± 2.1%) and Hyalomma scupense (1/185; 0.5 ± 1%). Mean overall tick infestation intensity was 7.4. Mixed infestations with two tick species were found in 10 sheep (23.3 ± 12.6%). All farmers (34/34) used ivermectin, and only 11% of them used acaricide to control ticks. The majority of R. bursa ticks (87/185; 45 ± 7%) were located on the ears 37/185; 20 ± 6%) and the testicles (50/185; 27 ± 6%). Giemsa-stained blood smears examination showed the presence of three haemopathogens: Anaplasma spp. (19/43; 44 ± 15%); Babesia spp. (6/43; 14 ± 10%) and Theileria spp. (1/43; 2 ± 4%). These results showed the presence of several low infestation burdens by ticks transmitting three haemopathogens.

Phenology and phylogeny of Hyalomma spp. ticks infesting one-humped camels (Camelus dromedarius) in the Tunisian Saharan bioclimatic zone.

In this study, we report the results of a survey of Hyalomma ticks infesting one-humped camels in southern Tunisia. Examinations were conducted every second or third month on 406 camels in Tataouine district from April 2018 to October 2019. A total of 1902 ticks belonging to the genus Hyalomma were collected. The ticks were identified as adult H. impeltatum (41.1%; n = 782), H. dromedarii (32.9%; n = 626), H. excavatum (25.9%; n = 493), and H. marginatum for a single specimen. Although the camels were infested by ticks throughout the year, the highest overall infestation prevalence was observed in April 2018 (p < 0.01). The overall infestation intensity varied between 2.7 and 7.4 ticks/animal. There were no statistically significant differences in tick infestation prevalence based on age categories of the camels, and the overall infestation prevalence was between 82.7% and 97.4%. Female camels were significantly more infested with ticks (88.3%) than males (65.5%) (p < 0.01). The infestation prevalence of camels varied significantly according to the region where sampling took place (p < 0.01), but no correlations were found with abiotic factors. The preferred attachment sites for adult Hyalomma ticks were the sternum (38.3%; n = 729/1902), around the anus (36.2%; n = 689/1902), udder (18.4%; n = 350/1902), and inner thigh (6.9%; n = 132/1902). Morphological classification of ticks was corroborated by sequencing the cytochrome c oxidase I (Cox1) and 16S rDNA genes, and these sequences were also used to infer phylogenetic relationships. A single H. dromedarii seemed to be a natural hybrid with H. rufipes. More attention should be devoted by the veterinary services to the infestation of camels by ticks.

TITLE: Phénologie et phylogénie des tiques Hyalomma spp. infestant les dromadaires (Camelus dromedarius) dans la zone bioclimatique saharienne tunisienne. ABSTRACT: Dans cette étude, les résultats d'une enquête concernant les tiques Hyalomma infestant les dromadaires dans le sud de la Tunisie sont présentés. Des examens ont été menés tous les deux ou trois mois sur 406 dromadaires dans le district de Tataouine entre avril 2018 et octobre 2019. Au total, 1902 tiques appartenant au genre Hyalomma ont été collectées. Les tiques adultes ont été identifiées comme H. impeltatum (41,1 % ; n = 782), H. dromedarii (32,9 % ; n = 626), H. excavatum (25,9 % ; n = 493) et un seul spécimen de H. marginatum. Bien que les dromadaires aient été infestés par les tiques tout au long de l'année, la prévalence globale d'infestation la plus élevée a été observée en avril 2018 (p < 0,01). L'intensité globale d'infestation variait entre 2,7 et 7,4 tiques/animal. Il n'y avait pas de différence statistiquement significative de l'infestation par les tiques en fonction des catégories d'âge des dromadaires, et la prévalence globale d'infestation se situait entre 82,7 et 97,4 %. Les dromadaires femelles étaient significativement plus infestés par les tiques (88,3 %) que les mâles (65,5 %) (p < 0,01). La prévalence d'infestation variait significativement en fonction des régions (p < 0.01) mais il n'y avait pas de corrélation avec les facteurs abiotiques. Les sites de fixation préférés des tiques Hyalomma adultes étaient le sternum (38,3 % ; n = 729/1902), autour de l'anus (36,2 % ; n = 689/1902), la mamelle (18,4 % ; n = 350/1902) et la face interne de la cuisse (6,9 % ; n = 132/1902). La classification morphologique a été corroborée par le séquençage des gènes de l'ADNr du cytochrome c oxydase I (Cox1) et du 16S, et ces séquences ont également été utilisées pour déduire les relations phylogénétiques. Un seul spécimen de H. dromedarii semblait être un hybride naturel avec H. rufipes. Une plus grande attention doit être accordée par les services vétérinaires quant à l'infestation des dromadaires par les tiques.

Relationships between specific antibody responses and clinical signs of dogs living in Tunisian endemic areas of canine leishmaniasis caused by Leishmania infantum.

The first step of the diagnostic process of canine leishmaniasis (CanL) is initiated by veterinarians and relies on their assessment of a high number of clinical signs common to other infectious diseases. We investigated herein the relationship between the clinical profile of 64 domestic dogs living in Tunisian endemic areas and their serological immune status with the aim to identify leishmanial serological markers of diagnosis and disease staging. Seven clinical signs were examined and a total clinical score that describes the number (TCS1) and the number plus the intensity of the clinical signs (TCS2) were determined. Laboratory tests consisted of parasitological examination (PE) of Giemsa-stained popliteal lymph node smears, indirect fluorescent antibody test (IFAT), IgG-, IgG1-, IgG2-Enzyme-Linked-Immunosorbent-Assay (ELISA), and IgG1-, IgG2- Western blotting (WB). Dogs' categorization according to the results of routine diagnostic tests, the TCS1 and TCS2, and the relative IgG1 and IgG2 specific reactivity allowed us to show that active CanL is characterized by an increased reactivity of the IgG2 specific antibodies. Interestingly, the IgG1 levels increased in parallel with the TCS1 and especially with the TCS2, indicating that this isotype is a better marker of dogs' health deterioration. PE & IFAT positive dogs which presented the highest TCS2 and IgG1 reactivity demonstrated significantly more severe weight loss and paleness of the mucosal membranes, suggesting that these signs characterize the latest stages of the disease. WB analysis showed that threeleishmanial polypeptides merit attention and further investigations. The antigens with MWs 32kDa reacting with IgG1 and 37kDa reacting withIgG2 antibodies were found associated with the results of diagnostic tests and late CanL stages, whereas the 24kDa antigen reacting with the IgG2 isotype and associated with low TCS2 seems to be a marker of the early stages.

Current status of tropical theileriosis in Northern Africa: A review of recent epidemiological investigations and implications for control.

Tropical theileriosis caused by the apicomplexan hemoparasite Theileria annulata is a tick-borne disease that constraints livestock production in parts of Europe, Asia and Africa. Four Hyalomma tick species transmit T. annulata in at least eight Africa countries (Mauritania, Morocco, Algeria, Tunisia, Egypt, Sudan, South Sudan and Ethiopia). The two dominant T. annulata vector ticks present in Africa, H. scupense and H. anatolicum, underlie two different patterns of transmission, which in turn greatly influence the epidemiology of tropical theileriosis. H. dromedarii and H. lusitanicum are also capable of transmitting T. annulata in North Africa, but their roles are associated with specific production systems and agro-ecological contexts. The emergence of resistance to the most widely used theilericidal compound, buparvaquone, continues to limit the effectiveness of chemotherapy. In addition, acaricide use is increasingly becoming unsustainable. Deployable T. annulata attenuated live vaccines established from local strains in Tunisia, Sudan and Egypt are available, and recent work has indicated that these vaccines can be protective under conditions of natural transmission. However, vaccination programmes may vary over space and time due to differences in the prevalence of disease amongst cattle populations, as well seasonal variation in vector activity. We review recent descriptive and analytical surveys on the epidemiology of T. annulata infection with reference to (a) demographic aspects such as breeds and ages of cattle herds previously exposed to distinct T. annulata infection pressures and (b) seasonal dynamics of tick activity and disease transmission. We then discuss how the wider endemic patterns that we delineate can underpin the development and execution of future vaccination programmes. We also outline options for integrated control measures targeting tick vectors and husbandry practices.

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

Leishmania (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.