Mourad Ben Said

First molecular survey and novel genetic variants' identification of Anaplasma marginale, A. centrale and A. bovis in cattle from Tunisia

Few data are available about the presence and distribution of Anaplasma species in cattle in North African countries. In this study prevalence, co-infections, risk factors and genetic diversity of Anaplasma species were evaluated in bovines from Northern Tunisia. A total of 232 cattle from 36 randomly selected farms in three Tunisian localities were investigated for the presence of Anaplasma species in blood by Real-time PCR and/or nested PCR. Overall infection rates of Anaplasma spp., Anaplasma marginale, Anaplasma centrale and Anaplasma bovis were 34.9%, 25.4%, 15.1%, and 3.9%, respectively. Anaplasma phagocytophilum was not detected in cattle. The most common co-infection pattern was an association of A. marginale and A. centrale (11.2%). Five cattle (2.1%) all reared in the sub-humid bioclimatic area, were co-infected by the three Anaplasma species. Molecular prevalence of Anaplasma infection varied significantly according to locality, bioclimatic area, tick infestation and type of breeding. Animals of the Holstein breed were less infected by A. marginale and A. centrale than other breeds. Genetic analysis of A. marginale msp4 gene indicated a high sequence diversity of Tunisian strains, suggesting a multiple introduction of infected cattle from different origins. Phylogenetic studies based on the 16S rRNA gene showed that the most prevalent A. centrale strains were closely related to the A. centrale vaccine strain. Moreover, all A. bovis variants clustered with other A. bovis sequences obtained from domestic and wild ruminant strains. This is the first molecular investigation on Anaplasma species in Tunisian cattle providing pivotal background for designing epidemiological studies and to develop control strategies in the country.

First molecular survey of Anaplasma bovis in small ruminants from Tunisia

To date, no information is available regarding the presence of Anaplasma bovis in the South Mediterranean area. In this study, prevalence, risk factors, and genetic diversity of A. bovis were assessed in small ruminants. A total of 563 healthy small ruminants (260 sheep and 303 goats), from 25 randomly selected flocks located in 5 localities from two bioclimatic areas in Tunisia, were investigated for the detection of A. bovis in blood by nested polymerase chain reaction (nPCR) assay. The overall infection rates of A. bovis were 42.7 and 23.8% in sheep and goats, respectively. Goats located in a sub-humid area were statistically more infected than those located in a humid area. A. bovis prevalence rate varied significantly according to sheep and goat flocks, and to the sheep breed. Infection with A. bovis was validated by sequencing. Sequence analysis based on the 16S rRNA gene showed that A. bovis from Tunisian goats and sheep clustered with other strain sequences detected from wild and domestic animals and published in GenBank. This study gives the first insight of presence of A. bovis DNA in small ruminants in Tunisia and suggests that these animal species may be playing an important role in the bovine anaplasmosis natural cycle caused by A. bovis in the South Mediterranean ecosystem.

Detection of novel strains genetically related to Anaplasma platys in Tunisian one-humped camels (Camelus dromedarius).

INTRODUCTION Little information is currently available regarding the presence of Anaplasma species in North African dromedaries. To fill this gap in knowledge, the prevalence, risk factors, and genetic diversity of Anaplasma species were investigated in Tunisian dromedary camels. METHODOLOGY A total of 226 camels from three different bioclimatic areas were sampled and tested for the presence of Anaplasma species by quantitative polymerase chain reaction (qPCR) and nested polymerase chain reaction (nPCR) assays. Detected Anaplasma strains were characterized by 16S rRNA sequence analysis. RESULTS Overall infection rate of Anaplasma spp. was 17.7%, and was significantly higher in females. Notably, A. marginale, A. centrale, A. bovis, and A. phagocytophilum were not detected. Animals were severely infested by three tick species belonging to the genus Hyalomma (H. dromedarii, H. impeltatum, and H. excavatum). Alignment, similarity comparison, and phylogenetic analysis of the 16S rRNA sequence variants obtained in this study suggest that Tunisian dromedaries are infected by more than one novel Anaplasma strain genetically related to A. platys. CONCLUSIONS This study reports the presence of novel Anaplasma sp. strains genetically related to A. platys in dromedaries from various bioclimatic areas of Tunisia. Findings raise new concerns about the specificity of the direct and indirect diagnostic tests routinely used to detect different Anaplasma species in ruminants and provide useful molecular information to elucidate the evolutionary history of bacterial species related to A. platys.

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.

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.

Anaplasma platys-like strains in ruminants from Tunisia

Molecular diagnosis of Anaplasma platys and related strains (A. platys-like) in carnivores and ruminants is challenging due to co-infections with cross-reacting strains, and require post-amplification sequencing of the hemi-nested PCR products traditionally generated by targeting the groEL gene. In this study, a Restriction Enzyme Fragment Length Polymorphism (RFLP) assay coupled to hemi-nested groEL PCR was developed to discriminate among A. platys and genetically related strains. This novel approach was used for investigating A. platys-like infection in 963 domesticated ruminants (241 goats, 355 sheep, and 367 cattle) from 22 delegations located in North Tunisia. Overall prevalence rates of A. platys-like were 22.8, 11, and 3.5% in goats, sheep, and cattle, respectively. Alignment, identity comparison, and phylogenetic analysis of the groEL sequence variants obtained in this study confirmed RFLP data suggesting that Tunisian ruminants are infected by novel unclassified Anaplasma strains genetically related to A. platys. Compared to sequencing, RFLP assay allows fast detection of A. platys and A. platys-like pathogens in the same sample and has a potential value especially when screening ticks, cats and ruminants, which can be a common host for these two bacteria. This newly developed molecular technique would provide valuable molecular tool for epidemiological studies related to A. platys as well as remove concern over specificity of serological and molecular methods routinely used to identify diverse Anaplasma strains and species in wild and domestic ruminants.

Hd86 mRNA expression profile in Hyalomma scupense life stages, could it contribute to explain anti-tick vaccine effect discrepancy between adult and immature instars?

Bm86 midgut protein has been used in order to control ticks of the Hyalomma genus. Previous studies demonstrated the inefficacity of this antigen in the control of Hyalomma scupense, whereas recombinant Hd86 antigen, the Bm86 ortholog in H. scupense produced in Pichia pastoris, was protective against larval H. scupense tick stage infestations but ineffective in the control of the adult stage. One possible explanation for this result is the variation in Hd86 expression levels between these two developmental stages. To test this hypothesis, Hd86 mRNA levels were characterized in H. scupense developmental stages. The expression profile of Hd86 demonstrated a significant variation between tick life stages and showed a significant reduction in the number of transcripts during feeding and, particularly after molting to adults. The most interesting result was noted after molting of engorged nymphs in unfed adults where the expression levels decreased significantly by 12.78 (10.77-17.39) (p<0.001) and 9.25 (5.77-15.72)-fold (p<0.001) in unfed males and unfed females, respectively. Comparing unfed nymphs to unfed adult ticks, the Hd86 expression levels decreased by 13.82 (5.39-24.45) (p=0.035) and 9.93 (2.87-22.08)-fold (p=0.038) in males and females respectively. Lower Hd86 mRNA levels in adult ticks should result in lower protein levels and thus less antibody-antigen interactions necessary for vaccine efficacy in ticks fed on vaccinated animals. Thus, the observed differences in Hd86 expression profile between immature and adult stages might explain, in part, the discrepancy of the Hd86 vaccine efficacy against these two life stages of H. scupense.

Spatio-temporal variations and genetic diversity of Anaplasma spp. in cattle from the North of Tunisia

In cattle, anaplasmosis is a tick-borne rickettsial disease caused by Anaplasma marginale, A. centrale, A. phagocytophilum, and A. bovis. To date, no information concerning the seasonal dynamics of single and/or mixed infections by different Anaplasma species in bovines are available in Tunisia. In this work, a total of 1035 blood bovine samples were collected in spring (n=367), summer (n=248), autumn (n=244) and winter (n=176) from five different governorates belonging to three bioclimatic zones from the North of Tunisia. Molecular survey of A. marginale, A. centrale and A. bovis in cattle showed that average prevalence rates were 4.7% (minimum 4.1% in autumn and maximum 5.6% in summer), 7% (minimum 3.9% in winter and maximum 10.7% in autumn) and 4.9% (minimum 2.7% in spring and maximum 7.3% in summer), respectively. A. phagocytophilum was not detected in all investigated cattle. Seasonal variations of Anaplasma spp. infection and co-infection rates in overall and/or according to each bioclimatic area were recorded. Molecular characterization of A. marginale msp4 gene indicated a high sequence homology of revealed strains with A. marginale sequences from African countries. Alignment of 16S rRNA A. centrale sequences showed that Tunisian strains were identical to the vaccine strain from several sub-Saharan African and European countries. The comparison of the 16S rRNA sequences of A. bovis variants showed a perfect homology between Tunisian variants isolated from cattle, goats and sheep. These present data are essential to estimate the risk of bovine anaplasmosis in order to develop integrated control policies against multi-species pathogen communities, infecting humans and different animal species, in the country.

Seasonal dynamics, spatial distribution and genetic analysis of Anaplasma species infecting small ruminants from Northern Tunisia

To date, there have been no reports on seasonal variations of Anaplasma spp. in South Mediterranean small ruminants. In this longitudinal field study, single and mixed Anaplasma spp. infections in small ruminants from five different governorates belonging to three bioclimatic zones from the North of Tunisia were evaluated according to seasons. A total of 1685 blood small ruminant samples were collected in spring (355 sheep and 241 goats), summer (249 sheep and 202 goats), autumn (236 sheep and 186 goats) and winter (132 sheep and 84 goats). Molecular survey of A. ovis and A. bovis showed that average prevalence rates were 35.6% (minimum 30.7% in spring and maximum 43.6% in autumn) and 7.4% (minimum 0.9% in spring and maximum 18.1% in summer), respectively, in sheep, and 46% (minimum 21.7% in summer and maximum 65.5% in winter) and 10.1% (minimum 2.2% in autumn and maximum 23.8% in summer), respectively, in goats. A. phagocytophilum was not detected in all investigated animals. The infection profiles of A. ovis and A. bovis show that anaplasmosis caused by A. ovis is endemic in small ruminants from all investigated bioclimatic areas during the four seasons but conversely, A. bovis infection is highly intensified only in the summer. A. ovis and A. bovis infections were validated by sequencing. The comparison of the 16S rRNA sequences of A. bovis variants showed 100% identity between Tunisian variants isolated from goats, sheep and cattle. The analysis of A. ovis msp4 sequences revealed two different genetic variants previously described in Italy. This is the first survey outlining seasonal dynamics of Anaplasma spp. infections in Tunisian small ruminants. This situation should to be taken into account if anaplasmosis control programs in these domesticated animals are envisaged.