First detection and molecular identification of Neospora caninum from naturally infected cattle and sheep in North Africa.

Neosporosis, caused by the protozoan Neospora caninum, is a major cause of reproductive failure in ruminants causing enormous economic losses. The objective of this study was to estimate the infection rate and molecular identification of N. caninum in Tunisian cattle and sheep. A total number of 348 meat samples were collected from 150 cows and 198 ewes slaughtered in the regional slaughterhouse of Béja (North-west Tunisia) and tested for the presence of N. caninum ITS1 gene using PCR 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. The overall molecular infection prevalence of N. caninum was significantly higher in cattle than in sheep (22 and 10.6%, respectively, p = .003). In sheep, the highest prevalence was observed in the northern Béja locality (31.2 ± 16.1), with the Noire de Thibar breed as the most infected sheep breed (31.7 ± 14.2%) (p < .001). In cattle, there were no differences in the molecular prevalence of N. caninum according to breeds and localities. The association between age and N. caninum molecular prevalence was statistically significant in both species; the highest prevalence was observed in sheep of more than one year of age (19.4 ± 9.1%), and in cattle between two and eight years of age (28.8 ± 10.9%). Comparison of the partial sequences of the ITS1 gene revealed 96%-100% similarity among our N. caninum amplicon and those deposited in GenBank. To our knowledge, this is the first detection and molecular identification of N. caninum in sheep and cattle in North Africa. This information is pertinent in designing control programmes that would reduce economic losses in the livestock industry.

First detection and molecular identification of Sarcocystis spp. in small ruminants in North-West Tunisia.

Sarcocystosis is a parasitic disease caused by varying Sarcocystis species infecting humans and animals. It is commonly found in small ruminants causing pathogenic effects. This contributes to detrimental economic loss for local farmers and the local economy due this disease. Although the distribution of Sarcocystis can be found all over the world, the species infecting small ruminants in Tunisia is still unknown. Through this study, we aim to estimate the molecular prevalence of natural infection with Sarcocystis spp. in sheep and goats using molecular identification. Also, phylogenetic analyses were used to identify the different species of this parasite infecting small ruminants in northern Tunisia for the first time. DNA was extracted from 198 and 121, sheep and goats meat samples, respectively. The molecular prevalence of Sarcocystis spp. in sheep and goats was 58.6% (116/198) and 50.4% (61/121), respectively. Compared to the Noire de Thibar and cross-breeds, the Barbarine sheep had the highest infection prevalence (63.4%) (p = .004). Five of the 116 positive samples were sequenced identifying Sarcocystis tenella from sheep. For goats, the sequencing results showed that five positive PCR products belonged to Sarcocystis capracanis species.

Molecular survey and genetic characterization of Anaplasma centrale, A. marginale and A. bovis in cattle from Algeria.

Bovine anaplasmosis could be caused by several Anaplasma species. The causative agents are transmitted by ticks and haematophagous arthropods with a high impact on both human and animal health. This study was conducted to estimate the infection rate and to characterize Anaplasma spp. in cattle from Algeria. A molecular survey was performed in Setif district (Northeast Algeria) where a total number of 180 cattle blood samples were collected and tested for the presence of Anaplasma spp. by PCR. Positive samples were genetically characterized based on the 16S rRNA and msp4 genes. PCRs revealed that the infection rates of Anaplasma spp., Anaplasma centrale, Anaplasma marginale and Anaplasma bovis were 42.2%; 39.4%; 11.1% and 4.4%, respectively. All tested animals were negative for A. phagocytophilum. Co-infection occurred in 10% (18/180) of the tested animals, and the most common co-infection pattern was an association between A. centrale and A. marginale (5.5%). Five cattle (2.7%) were co-infected by the three Anaplasma species. Holstein animals (58.1%) were more infected by A. centrale than the other breeds (p = .01). The molecular prevalence of A. centrale was significantly higher in males (54.2%) than in females (34.1%) (p = .001). A. marginale msp4 genetic analysis indicated a high sequence diversity of Algerian strains, suggesting the importation of live cattle from different origins. Phylogenetic analysis of the 16S rRNA gene of A. bovis and A. centrale revealed a low degree of genetic diversity. Our study suggests that different species of Anaplasma are simultaneously present in the Algerian cattle. To the best of our knowledge, this is the first molecular study and genetic characterization of Anaplasma spp. in Algerian cattle.

Molecular Study of Dirofilaria immitis and Dirofilaria repens in Dogs from Tunisia.

Dirofilaria immitis and Dirofilaria repens are mosquito-borne nematodes which infect primarily dogs as their main definitive hosts. They cause cardiopulmonary (D. immitis) or cutaneous (D. repens) dirofilariasis in canids and other carnivores and can accidentally be transmitted to humans where they can induce a variety of clinical outcomes depending on organ localization. Dirofilaria spp. infection in dogs was assessed using molecular methods (PCR and sequencing) to identify the different Dirofilaria species occurring in 200 dogs from Northern and Central Tunisia. The overall molecular prevalence of Dirofilaria spp. was 17.5% (35/200). The prevalence of D. immitis (14.5%) was significantly higher than for D. repens (3%). Molecular prevalence of D. immitis was significantly higher in suburban compared to urban and rural regions. There was no difference in molecular prevalence of D. immitis or D. repens according to the dogs' (sex or use). Dirofilaria immitis amplicons (accession numbers KR676386) fall into the same clade with D. immitis from China, India and Taiwan. Comparison of the partial sequences of D. repensITS2 rDNA gene (KR676387) revealed 99.6% similarity with D. repens reported in dogs from USA. It had also 97.6% similarity with D. repens from mosquitoes in Czech Republic. High dog parasite burdens should motivate both medical doctors and veterinarians to consider these frequent infections.

First Molecular Identification and Genetic Characterization of Theileria lestoquardi in Sheep of the Maghreb Region.

Theileria lestoquardi is the most prominent Theileria species in small ruminants that causes malignant theileriosis of sheep in Africa and Asia. In the present survey, blood samples and ticks were collected in Kebili (southern Tunisia) from 166 Queue Fine de l'Ouest sheep. Giemsa-stained blood smears, immunofluorescent antibody test (IFAT) and PCR were performed. The DNA was extracted from blood and analysed by PCR targeting 18S rRNA gene of Theileria spp. and then sequenced. A total number of 140 ticks were collected from a total number of 166 sheep during the four seasons. The ticks belonged to two genera and 4 species; the most frequent tick was Hyalomma excavatum 84.3% (118/140) and then Rhipicephalus spp. 15.7% (22/140). Only two animals had positive Giemsa-stained blood smears, and they were also positive by IFAT. The amplicons had 99.3 and 99.6% homology with the BLAST published T. lestoquardi amplicons. To our knowledge, this is the first report of T. lestoquardi in small ruminants within the Maghreb region.