Moez Mhadhbi

In vivo evidence for the resistance of Theileria annulata to buparvaquone

The 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.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes.

We developed a Leishmania infantum specific LAMP assay that was carried out using a set of, six primers targeting the cysteine protease B multi copy gene of L. infantum. Our result shows that we, successfully detect the L. infantum DNA and that amplification is specific as no cross reaction was seen, with L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii, Trypanosoma cruzi or, human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay, was higher with a detection limit of 50 fg/μl of genomic L. infantum parasite DNA. Accurate and rapid, diagnosis of canine leishmaniasis (CanL) is an important issue that allows early treatment and, prevents transmission. Our developed LAMP assay was used to evaluate occurrences of Leishmania infantum in seventy five (75) dogs from the field. Blood samples were used to perform LAMP assay, classical PCR, IFAT and microscopy that was used as gold standard. The IFAT in addition to, microscopy, are the basic techniques used for CanL diagnosis at the School of Veterinary Medicine, where we obtained our samples. Compared to molecular methods, the serology (IFAT) test shows the, best sensitivity (88.57%) with, however, a much lower specificity (52.5%) due to a relatively high, number of false-positive results (22 animals). The PCR assay shows a low sensitivity (37.14%) and, specificity around (82.5%). Our LAMP assay shows a suitable sensitivity (54%) and a good specificity, (80%), with however, positive (70%) and negative (66%) predictive values. Furthermore, the best, positive likelihood ratio (LR+) was obtained by LAMP assay (2.7). This technique presents the highest, kappa value (with a fair agreement of 0.34). Moreover, the relative stability of the reagents indicates, that LAMP may be a good alternative to a conventional PCR, especially under field conditions. Finally in, a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. This, is the first study that evaluates the L. infantum specific LAMP alongside other diagnostics tools for, CanL. Our results indicate a suitable sensitivity and specificity for the developed LAMP assay that could, has usefulness application on dogs and human L. infantum diagnosis.

Epidemiological study of sympatric Haemonchus species and genetic characterization of Haemonchus contortus in domestic ruminants in Tunisia.

Parasitic gastroenteritis caused by Haemonchus spp. is a major cause of economic losses in the livestock industry, especially in tropical and subtropical areas. The correct identification of various species, as well as knowledge regarding the epidemiology and genetic characterization of the principal circulating species, is essential for the establishment of sustainable control strategies. A study was carried out to determine the prevalence of Haemonchus species in sheep, goats and cattle slaughtered in Béja abattoir from January to June 2010 and also to analyse the genetic differences of Haemonchus contortus in these ruminants. During the study period 364, 271 and 152 abomasa of sheep, goats and cattle respectively, were examined showing overall prevalence rates of 17%, 33.6% and 7.23%, respectively. In addition, spicules morphometric study of 300 male worms randomly collected from sheep showed the presence of 239 (79.66%) H. contortus and 61 (20.33%) H. placei. Likewise, out of 508 adult male Haemonchus from goats, 325 (63.97%) H. contortus and 183 (36.02%) H. placei worms were identified. Whereas for cattle, out of 84 adult male Haemonchus, 52 (61.9%) H. contortus and 32 (38.09%) H. placei worms were identified. The study showed the association of H. contortus and H. placei as a predominant type of infection in all hosts, co-infection concerned 62.5% of sheep, 54.71% of goats and 37.5% of cattle. Using the polymerase chain reaction, the second Internal Transcribed Spacer region (ITS-2) of the nuclear ribosomal DNA (rDNA) of H. contortus was amplified and sequenced. A total of 16 ITS-2 sequences were identified (five from sheep, three from cattle and eight from goats). The 231 base pairs of different ITS-2 sequences were aligned and analysed. Distance based analysis using Neighbour-Joining method and parsimony analysis were used to construct phylogenetic trees to elucidate genetic relationships. The analyses categorized the ITS-2 sequence of H. contortus into four groups. Groups 1 and 4 were found exclusively in goats, whereas groups 2 and 3 were found in sheep and cattle. This study demonstrates variability in nucleotide sequence within the ITS-2 region that reveals genetic diversity among populations of H. contortus, including those from different domestic ruminant species in Tunisia. To our knowledge, this is the first work in North Africa describing the genetic diversity of H. contortus in domestic ruminants.

Prevalence of piroplasms in small ruminants in North-West Tunisia and the first genetic characterisation of Babesia ovis in Africa

In this study, the prevalence of piroplasms in sheep and goats was assessed with Giemsa-stained blood smear examination, PCR and nested PCR-restriction fragment length polymorphism (RFLP) to identify Babesia and Theileria species, respectively, in 338 small ruminants (172 sheep and 166 goats) from three sites in North-West Tunisia during the 2011 summer season. The overall infection prevalence of piroplasms in Giemsa-stained blood smears was 3.2% (11/338), with a parasitaemia ranging from 0.01 to 0.05%. PCR detected two species, namely Babesia ovis (in sheep and goats) and Theileria ovis (in sheep), with an overall prevalence of 16.3%. The molecular prevalence of B. ovis was significantly higher in sheep than in goats (17.4% and 9%, respectively, p = 0.034). The same trend was observed for T. ovis in sheep and goats (5.8% and 0%, respectively, p = 0.004). Comparison of the partial sequences of the 18S ssu rRNA gene revealed 100% similarity amongst Babesia from sheep and goats. The single Theileria sequence in this study showed 100% similarity to T. ovis. A high similarity with all the blasted genotypes was reported for Theileria and Babesia sequences. This is the first molecular detection of B. ovis and genetic characterisation of small ruminants’ piroplasms in Africa.

Sequence Polymorphism of Cytochrome b Gene in Theileria annulata Tunisian Isolates and Its Association with Buparvaquone Treatment Failure.

Background Buparvaquone (BW 720C) is the major hydroxynaphtoquinone active against tropical theileriosis (Theileria annulata infection). Previous studies showed that buparvaquone, similarly to others hydroxynaphtoquinone, probably acts by binding to cytochrome b (cyt b) inhibiting the electron transport chain in the parasite. Several observations suggested that T. annulata is becoming resistant to buparvaquone in many endemic regions (Tunisia, Turkey and Iran), which may hinder the development of bovine livestock in these areas. Methodology/Principal Findings In the present study we sought to determine whether point mutations in T. annulata cytochrome b gene could be associated to buparvaquone resistance. A total of 28 clones were studied in this work, 19 of which were obtained from 3 resistant isolates (ST2/12, ST2/13 and ST2/19) collected at different time after treatment, from a field treatment failure and nine clones isolated from 4 sensitive stocks of T. annulata (Beja, Battan, Jed4 and Sousse). The cytochrome b gene was amplified and sequenced. We identified five point mutations at the protein sequences (114, 129, 253, 262 and 347) specific for the clones isolated from resistant stocks. Two of them affecting 68% (13/19) of resistant clones, are present in the drug-binding site Q02 region at the position 253 in three resistant clones and at the position 262 in 11 out of 19 resistant clones. These two mutations substitute a neutral and hydrophobic amino acids by polar and hydrophilic ones which could interfere with the drug binding capabilities. When we compared our sequences to the Iranian ones, the phylogenetic tree analyses show the presence of a geographical sub-structuring in the population of T. annulata. Conclusions/Significance Taken together, our results suggest that the cytochrome b gene may be used as a tool to discriminate between different T. annulata genotypes and also as a genetic marker to characterize resistant isolates of T. annulata.

Hd86, the Bm86 tick protein ortholog in Hyalomma scupense (syn. H. detritum): Expression in Pichia pastoris and analysis of nucleotides and amino acids sequences variations prior to vaccination trials

The genus Hyalomma includes the most frequent tick species infesting livestock in North Africa, one of these species, Hyalomma scupense (syn. H. detritum) is particularly important due to its role in the transmission of tropical theileriosis to cattle (Theileria annulata infection). We have cloned and characterized the orthologs of the Bm86 gene from H. scupense strains collected over Tunisia in 2006 and 2009. The recombinant protein rHd86 was expressed in Pichia pastoris for vaccination purpose using a transcript from the 2006 strain. The rHd86 was then purified from the yeast culture supernatant by a filtration and a size exclusion process. It was recognized by specific anti-Bm86 antisera. An important extent of inter-specific diversity ranging from 35 to 40% was recorded between Hd86 and Bm86/Bm95 proteins whilst a very limited level of intra-specific diversity (1.7%) occurred between the Hd86 vaccine candidate protein and its homologues from H. scupense strains collected in 2009. These results emphasise the need for assessing the efficacy against H. scupense and others important cattle Hyalomma species in Tunisia of our Hd86 vaccine candidate alongside with a Bm86 vaccine.

Efficacy of Hyalomma scupense (Hd86) antigen against Hyalomma excavatum and H. scupense tick infestations in cattle

The Rhipicephalus microplus recombinant Bm86-based tick vaccines have shown their efficacy for the control of several Hyalomma cattle ticks genera, namely H. dromedarii and H. anatolicum. However, H. scupense species, the most important tick in North Africa has never been studied. Vaccination trials using either a recombinant Bm86-based vaccine or a recombinant Hd86-based vaccine (the Bm86 ortholog in H. scupense) were conducted in cattle against immature and adult H. scupense ticks and adult H. excavatum ticks. The results showed a 59.19% reduction in the number of scupense nymphs engorging on Hd86 vaccinated cattle. However, cattle vaccination with Bm86 or Hd86 did not have an effect on H. scupense or H. excavatum adult ticks infestations. These results showed that Hd86 vaccines are selectively effective against H. scupense immature instars and emphasize on an integrated anti-tick vaccine control in North Africa.

Population dynamics of ticks infesting the one-humped camel (Camelus dromedarius) in central Tunisia.

A tick population was monitored on 30 camels (Camelus dromedarius) over one year in Kairouan region, Central Tunisia. A total of 1630 ticks was collected and identified resulting in an estimate of different parasitological indicators. The ticks belonged to 2 genera and 5 species: Hyalomma impeltatum (53%) and Hyalomma dromedarii (45%) were the dominant species followed by Hyalomma excavatum (1%), Hyalomma marginatum (0.5%), and Rhipicephalus turanicus (0.5%) (p<0.001). Mean infestation prevalence was 90.6%; all the animals were infested by at least one tick from May to September. The highest mean prevalence was observed in H. impeltatum (60%), the lowest was reported in R. turanicus (0.03%) (p<0.05). Mean overall intensity of infestation was 4.4 ticks/animal. The highest mean intensity was observed in H. impeltatum (2.7 ticks/animal). Overall mean abundance of ticks was 4.4 ticks/animal. Different abiotic factors, namely monthly mean minimum and monthly mean maximum temperatures and the number of sunny days were positively correlated with overall monthly tick burdens which were in turn negatively correlated with the monthly mean relative humidity. This is the first study on camel tick dynamics in Tunisia.

Molecular characterization of Bm86 gene orthologs from Hyalomma excavatum, Hyalomma dromedarii and Hyalomma marginatum marginatum and comparison with a vaccine candidate from Hyalomma scupense

The ixodid ticks from the Hyalomma genus are important pests of livestock, having major medical and veterinary significance in Northern Africa. Beside their direct pathogenic effects, these species are vectors of important diseases of livestock and in some instances of zoonoses. Anti-tick vaccines developed in Australia and Cuba based on the concealed antigen Bm86 have variable efficacy against H. anatolicum and H. dromedarii. This variation in vaccine efficacy could be explained by the variability in protein sequence between the recombinant Bm86 vaccine and Bm86 orthologs expressed in different Hyalomma species. Bm86 orthologs from three Hyalomma tick species were amplified in two overlapping fragments and sequenced. The rate of identity of the amino acid sequence of Hm86, He86 and Hdr86, the orthologs of Bm86, respectively, in H. marginatum marginatum, H. excavatum and H. dromedarii, with the Bm86 proteins from Rhipicephalus microplus (Australia, Argentina and Mozambique) ranged between 60 and 66%. The obtained amino-acid sequences of Hmm86, He86 and Hdr86 were compared with the Hd86-A1 sequence from H. scupense used as an experimental vaccine. The results showed an identity of 91, 88 and 87% for Hmm86, He86 and Hdr86, respectively. A specific program has been used to predict B cells epitopes sites. The comparison of antigenic sites between Hd86-A1 and Hm86/Hdr86/He86 revealed a diversity affecting 4, 8 and 12 antigenic peptides out of a total of 28 antigenic peptides, respectively. When the Bm86 orthologs amplification protocol adopted in this study was applied to H. excavatum, two alleles named He86p2a1 and He86p2a2 were detected in this species. This is the first time that two different alleles of Bm86 gene are recorded in the same tick specimen. He86p2a1 and He86p2a2 showed an amino acid identity of 92%. When He86p2a1 and He86p2a2 were compared to the corresponding sequence of Hd86-A1 protein, an identity of 86.4 and 91.0% was recorded, respectively. When compared to He86, Hdr86 and Hm86, Bm86 used in commercial and experimental vaccines, showed a greater extent of diversity than noted when the same Hyalomma orthologs were compared to Hd86-A1. Although significant, these variations were less extensive within the Hyalomma genus. Accordingly, thus suggesting that Hd86-A1 vaccine candidate might be more appropriate to target Hyalomma tick species than corresponding Bm86 commercial vaccines. However, vaccination trials with both antigens are required to validate this hypothesis.

Engineering Attenuated Virulence of a Theileria annulata–Infected Macrophage

Live attenuated vaccines are used to combat tropical theileriosis in North Africa, the Middle East, India, and China. The attenuation process is empirical and occurs only after many months, sometimes years, of in vitro culture of virulent clinical isolates. During this extensive culturing, attenuated lines lose their vaccine potential. To circumvent this we engineered the rapid ablation of the host cell transcription factor c-Jun, and within only 3 weeks the line engineered for loss of c-Jun activation displayed in vitro correlates of attenuation such as loss of adhesion, reduced MMP9 gelatinase activity, and diminished capacity to traverse Matrigel. Specific ablation of a single infected host cell virulence trait (c-Jun) induced a complete failure of Theileria annulata–transformed macrophages to disseminate, whereas virulent macrophages disseminated to the kidneys, spleen, and lungs of Rag2/γC mice. Thus, in this heterologous mouse model loss of c-Jun expression led to ablation of dissemination of T. annulata–infected and transformed macrophages. The generation of Theileria-infected macrophages genetically engineered for ablation of a specific host cell virulence trait now makes possible experimental vaccination of calves to address how loss of macrophage dissemination impacts the disease pathology of tropical theileriosis.