Working towards the development of vaccines and chemotherapeutics against neosporosis-With all of its ups and downs-Looking ahead.

Neospora caninum is an apicomplexan and obligatory intracellular parasite, which is the leading cause of reproductive failure in cattle and affects other farm and domestic animals, but also induces neuromuscular disease in dogs of all ages. In cattle, neosporosis is an important health problem, and has a considerable economic impact. To date there is no protective vaccine or chemotherapeutic treatment on the market. Immuno-prophylaxis has long been considered as the best control measure. Proteins involved in host cell interaction and invasion, as well as antigens mediating inflammatory responses have been the most frequently assessed vaccine targets. However, despite considerable efforts no effective vaccine has been introduced to the market to date. The development of effective compounds to limit the effects of vertical transmission of N. caninum tachyzoites has emerged as an alternative or addition to vaccination, provided suitable targets and safe and efficacious drugs can be identified. Additionally, the combination of both treatment strategies might be interesting to further increase protectivity against N. caninum infections and to decrease the duration of treatment and the risk of potential drug resistance. Well-established and standardized animal infection models are key factors for the evaluation of promising vaccine and compound candidates. The vast majority of experimental animal experiments concerning neosporosis have been performed in mice, although in recent years the numbers of experimental studies in cattle and sheep have increased. In this review, we discuss the recent findings concerning the progress in drug and vaccine development against N. caninum infections in mice and ruminants.

New Nucleic Base-Tethered Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Compounds: Synthesis and Antiparasitic Activity.

Aiming toward compounds with improved anti-Toxoplasma activity by exploiting the parasite auxotrophies, a library of nucleobase-tethered trithiolato-bridged dinuclear ruthenium(II)-arene conjugates was synthesized and evaluated. Structural features such as the type of nucleobase and linking unit were progressively modified. For comparison, diruthenium hybrids with other type of molecules were also synthesized and assessed. A total of 37 compounds (diruthenium conjugates and intermediates) were evaluated in a primary screening for in vitro activity against transgenic Toxoplasma gondii tachyzoites constitutively expressing ß-galactosidase (T. gondii ß-gal) at 0.1 and 1 µM. In parallel, the cytotoxicity in non-infected host cells (human foreskin fibroblasts, HFF) was determined by alamarBlue assay. Twenty compounds strongly impairing parasite proliferation with little effect on HFF viability were subjected to T. gondii ß-gal half maximal inhibitory concentration determination (IC50) and their toxicity for HFF was assessed at 2.5 µM. Two promising compounds were identified: 14, ester conjugate with 9-(2-oxyethyl)adenine, and 36, a click conjugate bearing a 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl substituent, with IC50 values of 0.059 and 0.111 µM respectively, significantly lower compared to pyrimethamine standard (IC50 = 0.326 µM). Both 14 and 36 exhibited low toxicity against HFF when applied at 2.5 µM and are candidates for potential treatment options in a suitable in vivo model.

Synthesis, Photophysical Properties and Biological Evaluation of New Conjugates BODIPY: Dinuclear Trithiolato-Bridged Ruthenium(II)-Arene Complexes.

The synthesis, photophysical properties and antiparasitic efficacy against Toxoplasma gondii ß-gal (RH strain tachyzoites expressing ß-galactosidase) grown in human foreskin fibroblast monolayers (HFF) of a series of 15 new conjugates BODIPY-trithiolato-bridged dinuclear ruthenium(II)-arene complexes are reported (BODIPY=4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, derivatives used as fluorescent markers). The influence of the bond type (amide vs. ester), as well as that of the length and nature (alkyl vs. aryl) of the spacer between the dye and the diruthenium(II) complex moiety, on fluorescence and biological activity were evaluated. The assessed photophysical properties revealed that despite an important fluorescence quenching effect observed after conjugating the BODIPY to the diruthenium unit, the hybrids could nevertheless be used as fluorescent tracers. Although the antiparasitic activity of this series of conjugates appears limited, the compounds demonstrate potential as fluorescent probes for investigating the intracellular trafficking of trithiolato-bridged dinuclear Ru(II)-arene complexes in vitro.

First molecular detection of Neospora caninum from naturally infected slaughtered camels in Tunisia.

BACKGROUND: Neospora caninum has been documented to infect most domestic wildlife but is known to primarily infect dogs and cattle and is considered an important cause of abortion in camels. OBJECTIVE: The aim of this study was to estimate the molecular detection of Neospora caninum in tissues of naturally infected camelids. METHODS: Brain, tongue (bottom and tip) and masseter muscles from 35 slaughtered camelids from Tataouine and Médenine regions were collected (n = 140 samples). PCR was used to amplify and detect N. caninum DNA in tissues samples followed by sequencing of some PCR products. A phylogenetic tree was then constructed to compare the partial sequences of the ITS1 gene with GenBank sequences. Histopathology examination was used to detect Neospora spp. cysts, but no lesions were observed. RESULTS: The overall molecular detection of N. caninum in camelids was 34.3% (12/35). The highest molecular detection of N. caninum was recorded in animals of more than 3 years old (6/9) and in animals aged between 1 and 3 years old (4/12). Whilst, the lowest molecular detection (2/14) was observed in animals 1 year or younger (p = 0.035). There were no significant differences in molecular detection of N. caninum according to both locality and gender (p > 0.05). Similarly, there was no difference of prevalence between different anatomical locations. Comparison of the partial sequences of the ITS1 gene revealed 100-95.5% similarity among our N. caninum amplicon (MW551566) and those deposited in GenBank. CONCLUSION: These results highlight the presence of a risk infection by N. caninum in camels. For preventing N. caninum infection further studies are needed to improve our knowledge about the epidemiology of neosporosis in North Africa.

Molecular identification of Neospora caninum and co-infection with Toxoplasma gondii in genital apparatus of naturally infected cows in North Tunisia.

The aim of this study was to estimate the molecular prevalence of Toxoplasma gondii, Neospora caninum and the co-infection by both Apicomplexan parasites in uterus tissues of cows. PCR was used to detect T. gondii and N. caninum DNA in uterus from 140 uteri of slaughtered cows in the regional slaughterhouse of Béja (Northwest Tunisia). Positive PCR products were sequenced and used for the phylogenetic analysis. The overall molecular prevalence of T. gondii and N. caninum in cows' uterus was 5 and 15.57%, respectively. Co-infection prevalence by the two parasites was estimated to be 2.85%. Risk factors including the age categories significantly affected the molecular prevalence of T. gondii and N. caninum in cows' uterus. The highest molecular prevalence of T. gondii (11.5 ± 3.1) and N. caninum (21.1 ± 11.1; p = 0.038) was observed in cows aged of more than 8 years. There were no differences depicted according to cow's breeds and localities. Comparison of the partial sequences of the ITS1 gene revealed 100% similarity among our N. caninum sequence (MW136256) and those deposited in GenBank. The T. gondii sequence described in this study (MW260335) was 99.4-100% homologous to T. gondii sequences published in the GenBank.To the best of our knowledge, this is the first molecular evidence of N. caninum and T. gondii co-infection in naturally infected cows in North Africa. This information is pertinent in designing control programmes that would reduce economic losses in the livestock industry.

The quest of the best - A SAR study of trithiolato-bridged dinuclear Ruthenium(II)-Arene compounds presenting antiparasitic properties.

A structure activity relationship (SAR) study of a library of 56 compounds (54 ruthenium and 2 osmium derivatives) based on the trithiolato-bridged dinuclear ruthenium(II)-arene scaffold (general formula [(η6-arene)2Ru2(µ2-SR)3]+, symmetric and [(η6-arene)2Ru2(µ2-SR1)2(µ2-SR2)]+, mixed, respectively) is reported. The 56 compounds (of which 34 are newly designed drug candidates) were synthesized by introducing chemical modifications at the level of bridge thiols, and they were grouped into eight families according to their structural features. The selected fittings were guided by previous results and focused on a fine-tuning of the physico-chemical and steric properties. Newly synthesized complexes were characterized by NMR spectroscopy, mass spectrometry and elemental analysis, and four single-crystal X-ray structures were obtained. The in vitro biological assessment of the compounds was realized by applying a three-step screening cascade: (i) evaluation of the activity against Toxoplasma gondii RH strain tachyzoites expressing ß-galactosidase (T. gondii-ß-gal) grown in human foreskin fibroblast monolayers (HFF) and assessment of toxicity in non-infected HFF host cells; (ii) dose-response assays using selected compound, and (iii) studies on the effects in murine splenocytes. A primary screening was performed at 1 and 0.1 µM, and resulted in the selection of 39 compounds that inhibited parasite proliferation at 1 µM by more than 95% and reduced the viability of HFF by less than 49%. In the secondary screening, dose-response assays showed that the selected compounds exhibited half maximal inhibitory concentration (IC50) values for T. gondii-ß-gal between 0.01 µM and 0.45 µM, with 30 compounds displaying an IC50 lower than 0.1 µM. When applied to non-infected HFF monolayers at 2.5 µM, 8 compounds caused more than 90% and 31 compounds more than 30% viability impairment. The tertiary screening included 14 compounds that did not cause HFF viability loss higher than 50% at 2.5 µM. These derivatives were assessed for potential immunosuppressive activities. First, splenocyte viability was assessed after treatment of cells with concanavalin A (ConA) and lipopolysaccharide (LPS) with compounds applied at 0.1 and 0.5 µM. Subsequently, the 5 compounds exhibiting the lowest splenocyte toxicity were further evaluated for their potential to inhibit B and T cell proliferation. Overall, compound 55 [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-o-CF3)2(µ2-SC6H4-p-OH)]Cl exhibited the most favorable features, and will be investigated as a scaffold for further optimization in terms of anti-parasitic efficacy and drug-like properties.

Conjugates Containing Two and Three Trithiolato-Bridged Dinuclear Ruthenium(II)-Arene Units as In Vitro Antiparasitic and Anticancer Agents.

The synthesis, characterization, and in vitro antiparasitic and anticancer activity evaluation of new conjugates containing two and three dinuclear trithiolato-bridged ruthenium(II)-arene units are presented. Antiparasitic activity was evaluated using transgenic Toxoplasmagondii tachyzoites constitutively expressing ß-galactosidase grown in human foreskin fibroblasts (HFF). The compounds inhibited T.gondii proliferation with IC50 values ranging from 90 to 539 nM, and seven derivatives displayed IC50 values lower than the reference compound pyrimethamine, which is currently used for treatment of toxoplasmosis. Overall, compound flexibility and size impacted on the anti-Toxoplasma activity. The anticancer activity of 14 compounds was assessed against cancer cell lines A2780, A2780cisR (human ovarian cisplatin sensitive and resistant), A24, (D-)A24cisPt8.0 (human lung adenocarcinoma cells wild type and cisPt resistant subline). The compounds displayed IC50 values ranging from 23 to 650 nM. In A2780cisR, A24 and (D-)A24cisPt8.0 cells, all compounds were considerably more cytotoxic than cisplatin, with IC50 values lower by two orders of magnitude. Irrespective of the nature of the connectors (alkyl/aryl) or the numbers of the di-ruthenium units (two/three), ester conjugates 6-10 and 20 exhibited similar antiproliferative profiles, and were more cytotoxic than amide analogues 11-14, 23, and 24. Polynuclear conjugates with multiple trithiolato-bridged di-ruthenium(II)-arene moieties deserve further investigation.

Coumarin-Tagged Dinuclear Trithiolato-Bridged Ruthenium(II)⋠Arene Complexes: Photophysical Properties and Antiparasitic Activity.

The synthesis, characterization, photophysical and biological properties of 13 new conjugate coumarin-diruthenium(II)⋅arene complexes against Toxoplasma gondii are presented. For all conjugate organometallic unit/coumarins, an almost complete loss of fluorescence efficacy was observed. However, the nature of the fluorophore, the type of bonding, the presence and length of a linker between the coumarin dye and the ruthenium(II) moiety, and the number of dye units influenced their biological properties. The in vitro activity against a transgenic T. gondii strain grown in human foreskin fibroblasts (HFF) leads to IC50 values for T. gondii ß-gal from 105 to 735 nM. Of note is that nine compounds displayed lower IC50 than the standard drug pyrimethamine. One compound applied at its IC50 did not affect B-cell proliferation but had an impact on T-cell proliferation in murine splenocyte cultures. Transmission electron microscopy of T. gondii ß-gal-infected HFF showed that treatment predominantly affected the parasites' mitochondrion.

Neospora caninum Natural Infection in Tunisian Rams: Serological Study and Molecular Identification of Infection in Semen.

PURPOSE: The aim of this study was to estimate the seroprevalence and molecular prevalence and perform a molecular identification of Neospora caninum in semen of Tunisian rams. METHODS: A total of 92 blood samples were collected from four farms located in four Tunisian governorates (Jendouba, Kairouan, Zaghouan and Ben Arous) and samples were screened with a commercial ELISA kit for N. caninum antibodies. For the same rams, semen samples were collected and tested for the presence of N. caninum ITS1 gene using PCR. Five amplicons were randomly selected for sequencing. A phylogenetic tree was constructed to compare the partial sequences of the ITS1 gene with sequences deposited in GenBank. RESULTS: The seroprevalence of N. caninum infection was 25% (23/92) and PCR revealed that the molecular infection prevalence in semen was 11.95% (11/92). Kappa test showed an average agreement between seroprevalence and parasite prevalence in semen (κ = 0.44). The highest molecular prevalence was for rams that accomplished more than two mating seasons (21.0 ± 12.1%) compared to those performed less than two mating seasons and yearling individuals (4.0 ± 5.5%) (P = 0.01). There were no differences in N. caninum molecular prevalence according to either breed or locality. Comparison of the partial sequences of the ITS1 gene revealed 99-100% similarity with those deposited in GenBank. CONCLUSION: To the best of our knowledge, this is the first detection and molecular identification of N. caninum in semen from rams in North Africa. Our findings indicate that N. caninum infection rate was high in rams.

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.

First molecular detection and phylogenetic analysis of Neospora caninum DNA from naturally infected goats in Northwest Tunisia.

Neospora caninum is an intracellular protozoan parasite from the phylum Apicomplexa, mainly associated with abortions and causing enormous economic losses. We aimed, by the present study, to estimate the molecular prevalence and phylogenetic analyses of natural infection with N. caninum in Tunisian goats. A total number of 121 meat samples were collected from slaughtered goats in the regional slaughterhouse of Béja (Northwest Tunisia) and tested from N. caninum ITS1 gene using PCR followed by sequencing of PCR products. Phylogenetic analyses were used to identify this parasite infecting goats in Nortwest Tunisia. The overall molecular prevalence was 19% (23/121). The highest molecular prevalence of N. caninum was observed in goats aged between 2 and 4 years (31.9 ± 13.27%) (P = 0.004). There was no difference in the overall molecular prevalence of N. caninum according to both localities and animal breeds. Comparison of the partial sequences of the ITS1 gene revealed 99-100% similarity with GenBank sequences. A high similarity with all the blasted genotypes was reported for N. caninum sequences. This is the first molecular study and genetic characterisation of N. caninum in North African goats.

Molecular detection and phylogenetic analyses of Toxoplasma gondii from naturally infected sheep in Northern and Central Tunisia.

Toxoplasmosis is a worldwide zoonosis caused by the parasitic protozoan Toxoplasma gondii. It can infect all warm-blooded vertebrate species and causes abortions and birth defects in pregnant women and pregnant ewes. The objective of this study was to estimate the prevalence of infection with T. gondii in sheep meat in the region of Sidi Bouzid (central Tunisia) and Beja (northern Tunisia), the realization of a descriptive study of risk factors and the phylogenetic analyses of T. gondii. Neck muscle samples were obtained from 174 ewes and ewe lamb slaughtered in Sidi Bouzid and 150 lambs slaughtered in Beja. DNA was extracted from the samples using the Wizard® genomic DNA purification kit. A nested PCR using two pairs of primers (NN 1 and NN2, Tg-NP1 and Tg-NP2) were used to detect infection with T. gondii, which was then confirmed by sequencing. Eight T. gondii amplicons were sequenced (accession number KT896498) and deposited in GenBank. The T. gondii amplicons showed 97-100% identities with GenBank sequences. A phylogenetic tree was then constructed. The nested PCR detected T. gondii DNA in 31% of animals tested in Sidi Bouzid and 32% of lambs tested in Beja. No significant difference in the prevalence of T. gondii infection was established between the two tested regions. In both regions, no significant variation of the infection depending on age, breed and locality was found.

Molecular detection of Toxoplasma gondii infection in slaughtered ruminants (sheep, goats and cattle) in Northwest Tunisia.

The present study aimed to estimate the molecular prevalence of T. gondii infection in meat from slaughtered sheep, goats and cattle in Northwest Tunisia (Béja district). PCRs were performed on genomic DNA extracted from 420 meat samples (150 ewes, 120 goats and 150 cows). The overall molecular prevalence of T. gondii in sheep, goats and cattle were 33.3 (50/150), 32.5 (39/120) and 19.3% (29/150), respectively. Toxoplasma gondii molecular prevalences in the three meat ruminant species were significantly higher in adults compared to young animals (p<0.001). The infection prevalence differed significantly within localities in sheep (p<0.001), goats (p<0.001) and cattle (p=0.019). These results provide important information about exposure of humans to T. gondii through the consumption of raw or undercooked ruminants' meat. Extension programmes should be implemented to decrease the risk of infection related to sheep, goats and cattle meat manipulation and raw or undercooked meat consumption.

First molecular detection and characterization of Sarcocystis species in slaughtered cattle in North-West Tunisia.

Sarcocystis spp. is one of the most prevalent foodborne parasites infecting both animals and humans. Consumption of raw or undercooked infected meat is a risk factor of human intestinal sarcocystosis. The present study aimed to estimate the prevalence of Sarcocystis species infecting slaughtered Tunisian cattle in North-West Tunisia (Béja governorate). DNA was extracted from 150 beef meat samples and a PCR-restriction fragment length polymorphism was used for identification. The overall infection prevalence of Sarcocystis spp. was 38% (57/150). Two species were identified, namely S. hominis (25%; 39/150) and S. cruzi (12%; 18/150). For both species, the highest prevalence was in Thibar locality (52.9 and 17.6% for S. hominis and S. cruzi, respectively). The molecular prevalence of S. cruzi was significantly higher in animals aged between two and eight years (19.2%; 10/52). This is the first molecular identification of Sarcocystis species in Tunisian cattle. Further studies in both human and animal Tunisian populations are needed to rank this parasitic disease among others.

Infestation of the spur-thighed tortoise (Testudo graeca) by Hyalomma aegyptium in Tunisia.

We examined 210 spur-thighed tortoises (Testudo graeca) for the presence of ticks in Tunisia during May 2014. A total number of 602 adult ticks were collected and identified leading to the estimation of parasitological indicators. All the ticks belonged to a single species: Hyalomma aegyptium. The mean infestation prevalence was 66.2%, mean overall infestation intensity and abundance were 4.33 and 2.86 ticks/tortoise respectively. Our survey showed that tortoises were significantly more infested by male ticks than females (p<0.001). The ticks were mainly present in the posterior limbs compared to other body regions (p<0.05). There was no significance variation of length and weight of tortoises according to sex (p<0.05). There was a significant correlation between the tortoises' size (length and weight) and tick infestation. This study showed high tick burdens of spur-thighed tortoises in Tunisia; further investigations are needed to determine exactly the role of this tick species in the transmission of different zoonotic pathogens.