Mohamed Aziz Darghouth

Innate and Adaptive Immune Responses Co-operate to Protect Cattle against Theileria annulata

For many years it was assumed that Theileria annulata resembled T. parva, parasitizing lymphocytes and causing lymphoproliferative disease, with the two species being controlled by similar protective immune responses. Patricia Preston et al. here review the evidence that has led to a different view of T. annulata. It is now thought that the schizonts of T. annulata inhabit macrophages and B cells, and that tropical theileriosis is not a lymphoproliferative disease. Both innate and adaptive responses contribute to recovery from infection and resistance to challenge and cytokines produced by infected and uninfected cells influence the outcome of infection. Partial protection has been stimulated recently by defined recombinant antigens; efficacy depended upon the delivery system.

A preliminary study on the attenuation of Tunisian schizont-infected cell lines of Theileria annulata

Abstract Four Theileria annulata cell lines were characterised at low passage levels using two polymorphic markers and then used to infect calves. Their virulence seemed to be related to the number of genotypes present within the cell line. In all, 3 of the 4 cell lines were cultured up to passage 100 or 200 and inoculated into calves. Their characterisation using the same markers indicated that the attenuation was related to a reduction in the parasite polymorphism down to a single genotype. The immunogenicity of the three attenuated cell lines was assessed in calves using two types of challenge. Optimal protection was observed against homologous challenges. The level of immunity to heterologous challenges appeared to decrease with attenuation and seemed to depend on the cell line used.

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.

Mechanism(s) of attenuation of Theileria annulata vaccine cell lines.

Summary Attenuated vaccines are an important means of controlling Theileria annulata infection of cattle. Production is by prolonged cultivation of macroschizont‐infected cells. The mechanism of attenuation remains unclear. There are three general nonmutually exclusive possibilities: Selection of avirulent subpopulations, genome rearrangements and alterations in gene expression. Several groups, including ours, have provided evidence that the population structure usually tends to simplify during attenuation. Our data on the T. annulata (Ta) Ankara cell line show that attenuation is not necessarily accompanied by the population becoming clonal. We have been unable to detect large DNA rearrangements. Evidence for alterations in host and parasite gene expression during attenuation is available. With respect to the host we have shown that attenuation is accompanied by loss of expression of parasite induced matrix metalloproteinases (MMPs). However, in different lines different protease activities are involved. In the T. annulata Ode line we have shown that 8 activities (including MMP9) are downregulated and that this correlates with a loss of metastatic behaviour. This has previously been shown in vitro using reconstituted basement membrane (Matrigel™) and is demonstrated in vivo using scid mice in this study. Thus part of the pathology, namely the ability to disseminate, mediated by host MMPs, is lost upon attenuation. Re‐isolation experiments have shown that the reduction/loss of MMP is a stable transferable trait. A logical extension is that loss of MMP activity (and virulence in general) must be at the most fundamental level a genetic trait of the parasite. Evidence for loss of parasite gene expression is implied by the loss of the ability to differentiate into merozoites on attenuation. Specific evidence for loss of parasite gene expression has been obtained using differential RNA display. We view virulence as a multifactorial phenomenon involving interacting subpopulations of cells and attenuation is a threshold effect whereby the number of virulence factors is reduced below a critical level. On this basis there will be many different ways to achieve attenuation.

Cattle infestation by Hyalomma ticks and prevalence of Theileria in H. detritum species in Tunisia.

Seventy-four cattle, from three farms endemic for tropical theileriosis in the north of Tunisia, were studied for tick populations from June 1991 to June 1992. Ticks were removed from cattle twice a month in the summer and every month the rest of the year. They were identified and assessed for Theileria infection. A total of 5083 Hyalomma adult ticks were collected and the major species found was H. detritum (84.3%). The activity of this species is limited between June and August with a peak in numbers observed at the end of June and the beginning of July. Amongst the 2356 Hyalomma ticks dissected, no evidence of salivary gland infection was found in either H. m. marginatum or H. a. excavatum. However, 12.4% (277/2230) of H. d. detritum dissected ticks were infected with Theileria species and amongst these, 62% had one to two sporoblasts in their salivary glands (range 1-91). The prevalence, but not the intensity, of infection was greater in females than in male ticks, and the cases of tropical theileriosis followed the peak of infected females. This suggests that female ticks have a more important role in theileriosis transmission than male ticks. A significantly lower number of adult H. detritum were collected from calves, at their first tick season, than from adult cattle. Finally, this study showed that the infestation level of cattle by H. d. detritum and the prevalence of Theileria-infection in these cattle varied between the three farms studied.

Epidemiology of tropical theileriosis (Theileria annulata infection of cattle) in an endemic region of Tunisia: characterisation of endemicity states

A serological survey on tropical theileriosis was conducted on a sample of 54 farms in a region within the semi-arid bioclimatic zone of Tunisia. Screening of cattle sera at a dilution of 1/160 using the indirect immunofluorescent antibody test with the schizont antigen of Theileria annulata, revealed the presence of animals with positive sera in 92.15% of the sampled farms. The exposure of calves to infection in the first season was shown to be significantly lower than in older cattle. Three endemic situations were identified based on the serological profiles of herds and the incidence and age distribution of disease cases. Endemic stability was observed in farms showing a sero-prevalence of 100% in cattle of four theileriosis seasons or more and by the incidence of the highest disease levels in cattle at their second and third theileriosis season. High endemic instability was identified on the basis of low sero-prevalence rates and the occurrence of the highest disease incidence in cattle at fourth theileriosis season or more.

Population diversity and multiplicity of infection in Theileria annulata

Graphical abstract Research highlights ► Natural populations of Theileria annulata show extensive diversity. ► Evidence of panmixia with linkage disequilibrium detected in some localities. ► Individual cattle are infected with a number of genotypes. ► Multiplicity of infection positively correlates with age of host.

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.

Review on the experience with live attenuated vaccines against tropical theileriosis in Tunisia: Considerations for the present and implications for the future

The choice of a vaccine candidate against tropical theileriosis was based on key epidemiological considerations inherent to the main target endemic situation in Tunisia. One of the attenuated cell lines was retained as a vaccine candidate on the basis of small-scale trials testing the intrinsic safety aspects of the vaccine for the targeted animal categories in the dominant endemic situation where it will be used. The vaccine efficacy was confirmed in pilot vaccination campaigns using fresh culture and thawed cryopreserved vaccine doses delivered to the field using a new delivery system not requiring a cold chain. The potential benefit of the live attenuated Tunisian vaccine was evaluated using simple economical models.

In vitro evidence that the pastoral Artemisia campestris species exerts an anthelmintic effect on Haemonchus contortus from sheep

Occurrence of anthelmintic resistant strains of helminths is increasing. The aim of this study was to evaluate the in vitro anthelmintic activity of Artemisia campestris in comparison to albendazole against Haemonchus contortus of sheep. In this respect, in vitro anthelmintic activities of crude aqueous and crude ethanolic extracts of aerial parts of A. campestris were investigated on eggs and adults of Haemonchus contortus. Chemical analyses revealed that overall profile of both extracts samples were dominated by flavonoids among them quercetin and apigenin derivatives were the most abundant phenolics constituents. Both extract types completely inhibited egg hatching at a concentration close to 2 mg/ml. Lethal concentration 50% of A. campestris ethanolic and aqueous extracts were 0.83 and 1.00 mg/ml respectively (p < 0.05). The ethanolic extract showed better in vitro activity against adult parasites than the aqueous extract in terms of the paralysis and/or death of the worms at different hours post-treatment. Dose dependent activity was also observed for both extract. After 8 and 24 h of exposure, the ethanolic extract induced 91.3 and 100% mortality at the highest tested concentration respectively, while the aqueous extract induced 3.22 and 70.96% at the same concentration respectively.To our knowledge, these results depict for the first time that A. campestris possesses in vitro anti-Haemonchus contortus properties.