Repeated Cross-Sectional Survey of Ectoparasites in Sheep from Central Tunisia: Does Low Prevalence Indicate Good Hygiene or Resistance to Ectoparasites?

Sheep ectoparasites such as chewing lice, fleas and ticks are serious constraints to sheep productivity and are the cause of skin lesions in animals that decrease their market value. This study aims at investigating the ectoparasite fauna infesting small ruminants in the district of Sidi Bouzid (central Tunisia). A total of 1243 Barbarine and Queue Fine de l'Ouest (QFO) sheep were examined every two months for one year. Of the total animals examined, 74 were infested by at least 1 parasite group (5.95%). Three ectoparasite groups were identified as Psoroptes ovis (0.48%; 6/1243), ticks (5.3%; n = 66/1243) and one specimen of Ctenocephalides canis (0.08%; n = 1/1243). The most abundant tick among the 358 specimens was Rhipicephalus sanguineus sensu lato (n = 337; 94.1%), followed by Hyalomma impeltatum (n = 7/358; 1.9%), H. dromedarii (n = 7/358; 1.9%), H. excavatum (n = 5/358; 1.4%) and only two specimens of H. scupense (n = 2/358; 0.55%). The sheep herds showed low infestation prevalence by ectoparasite over the year, with a significant difference according to the seasons (p < 0.05). A higher infestation prevalence was recorded in March (14.36%). Barbarine sheep breed showed significantly higher infestation prevalence (16.8%) compared to QFO (0.8%) (p < 0.01). There were no differences in infestation prevalence according to sex of the animal or age groups. Knowledge of the ectoparasite population harboured by sheep, its activity dynamics and risk factors is required to develop effective ectoparasite control options. The low prevalence of ectoparasite infestation in sheep reported here may be due to possible genetic resistance or simply to successful hygiene measures implemented by farmers.

Development and field evaluation of PCR assays based on minimum length Bm86 cDNA fragments required for Rhipicephalus and Hyalomma tick species delineation.

Introduction: Hyalomma and Rhipicephalus ticks are important genera that can transmit diseases to both animals and humans, including Crimean-Congo hemorrhagic fever, tick-borne encephalitis, and several types of spotted fever. The accurate identification of tick species is essential for the effective control and prevention of tick-borne diseases. However, traditional identification methods based on morphology can be challenging and subjective, leading to errors. The development of DNA markers has provided more precise and efficient methods for tick species identification, but the currently available markers have limitations in their discriminatory power and sensitivity. To address this need for more sensitive and specific markers, this study aimed to identify two minimum sequence fragments required for tick Hyalomma and Rhipicephalus species identification using the Bm86 cDNA marker, which has previously been shown to be in perfect agreement with the current taxonomy of hard ticks based on its complete sequence. Methods: Based on our in silico determination that a minimum sequence of 398 bp for Rhipicephalus spp. (from 1487 to 1884) and 559 bp for Hyalomma species (from 539 to 1097) was necessary for species delineation, two distinct PCR assays were developed to apply these sequences in practice. Results and discussion: Discrimination between species within each genus was achieved through sequence homology and phylogenetic analysis following the sequencing of the two PCR products. Subsequently, their performance was evaluated by testing them on the field-collected ticks of the Hyalomma and Rhipicephalus genera obtained from various host animals in different geographic regions of Tunisia. The use of shorter partial sequences specific to the tick genera Rhipicephalus and Hyalomma, which target the tick's RNA banks, could represent a significant advance in the field of tick species identification, providing a sensitive and discriminatory tool for interspecific and intraspecific diversity analysis.

Cattle co-infection patterns by hemopathogens and their phylogenetic analysis during the tick season in Constantine and Mila, Northeast Algeria.

Tropical theileriosis, babesiosis, and anaplasmosis are the most dominant tick-borne infections in North Africa where they cause significant economic losses in ruminants' industry. The aim of the present work was to study infections and co-infection patterns in 66 cattle with clinical signs of piroplasmosis and/or anaplasmosis in two localities, Beni Hamidene and Grarem Gouga, districts of Constantine and Mila (Northeast of Algeria), respectively. This study was conducted between early May and late September during four years 2017, 2018, 2020, and 2021. PCR showed that the most frequent pathogen in cattle with clinical signs of piroplasmosis and/or anaplasmosis was Theileria annulata (66/66; 100%) followed by Babesia bovis (21/66; 31.8%), Anaplasma marginale (15/66; 22.7%), and Babesia bigemina (3/66; 4.5%) (p < 0.001). Giemsa-stained blood smears examinations revealed that 66.7% (44/66); 10.6% (7/66); and 9.1% (6/66) of cattle were infected by T. annulata, Babesia spp., and A. marginale, respectively (p < 0.001). PCR revealed seven co-infection patterns: T. annulata/A. marginale (15/66; 22.7%), T. annulata/B. bovis (21/66; 31.8%), T. annulata/B. bigemina (3/66; 4.5%), T. annulata/A. marginale/B. bovis (7/66; 10.6%), T. annulata/B. bovis/B. bigemina (2/66; 3%), T. annulata/A. marginale/B. bigemina (1/66; 1.5%), and T. annulata/A. marginale/B. bigemina/B. bovis (1/66; 1.5%). Phylogenetic analyses showed that T. annulata Tams1 and B. bigemina gp45 sequences were identical to isolates from Mauritania and South Africa, respectively. The three A. marginale amplicons obtained herein had 99.63 to 99.88% similarity between them. This study provides data that can be used to improve control programs targeting these cattle hemopathogens.

Diversity and Phylogeny of Cattle Ixodid Ticks and Associated Spotted Fever Group Rickettsia spp. in Tunisia.

Tick-borne rickettsioses are mainly caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the Rickettsia genus. So far, the causative agents of SFG rickettsioses have not been detected in cattle ticks from Tunisia. Therefore, the aim of this study was to investigate the diversity and phylogeny of ticks associated with cattle from northern Tunisia and their associated Rickettsia species. Adult ticks (n = 338) were collected from cattle in northern Tunisia. The obtained ticks were identified as Hyalomma excavatum (n = 129), Rhipicephalus sanguineus sensu lato (n = 111), Hyalomma marginatum (n = 84), Hyalomma scupense (n = 12) and Hyalomma rufipes (n = 2). After DNA extraction from the ticks, 83 PCR products based on the mitochondrial 16S rRNA gene were sequenced and a total of four genotypes for Rh. sanguineus s.l., two for Hy. marginatum and Hy. excavatum and only one for Hy. scupense and Hy. rufipes were recorded, with the occurrence of one, two and three novel genotypes, respectively, for Hy. marginatum, Hy. excavatum and Rh. sanguineus s.l. mitochondrial 16S rRNA partial sequences. The tick DNA was tested for the presence of Rickettsia spp. by using PCR measurements and sequencing targeting three different genes (ompB, ompA and gltA). Of the 338 analyzed ticks, 90 (26.6%), including 38 (34.2%) Rh. sanguineus s.l., 26 (20.1%) Hy. excavatum, 25 (29.8%) Hy. marginatum and one (50%) Hy. rufipes tick, were positive for Rickettsia spp. Based on 104 partial sequences of the three analyzed genes, the BLAST analysis and phylogenetic study showed the infection of Hy. excavatum, Hy. marginatum and Rh. sanguineus s.l. tick specimens with R. massiliae, R. aeschlimannii and R. sibirica subsp. mongolitimonae and one Hy. rufipes tick specimen with R. aeschlimannii. In addition, coinfection with R. massiliae and R. aeschlimannii was reported in one Hy. marginatum and one Rh. sanguineus s.l. tick specimen, while a coinfection with R. massiliae and R. sibirica subsp. mongolitimonae was recorded in one Rh. sanguineus s.l. tick specimen. In conclusion, our study reports, for the first time in Tunisia, the infection of cattle ticks belonging to Hyalomma and Rhipicephalus genera with zoonotic Rickettsia species belonging to the SFG group.

Phytochemical profile and anthelmintic effects of Laurus nobilis essential oil against the ovine nematode Haemonchus contortus and the murine helminth model Heligmosomoides polygyrus.

Small ruminant production in tropical and temperate countries faced substantial anthelmintic resistance due to the intensive use of commercial anthelmintic drugs. Therefore, alternative treatments including natural bioactive compounds with anthelmintic potential have been investigated looking for its successfully use in the parasite control. In the present study, we describe the chemical profile of Laurus nobilis essential oil (EO), the in vitro anthelmintic activity of L. nobilis EO against Haemonchus contortus and its in vivo anthelmintic effect against the murine helminth parasite model Heligmosomoides polygyrus. Chromatographic profile of L. nobilis (EO) extracted from the leaves of L. nobilis have shown the presence of monterpens 1,8-cineol (Eucalyptol) (29.47%), D-Limonène (18.51%) and Linalool (10.84%) in high fractions. The in vitro anthelmintic potential was expressed by an ovicidal effect against H. contortus egg hatching with inhibition value of 1.72 mg/mL and 87.5% of immobility of adult worms after 8 h of exposure to 4 mg/mL of L. nobilis EO. Regarding, the in vivo anthelmintic potential, L. nobilis (EO) at 2400 mg/kg bw completely eliminated the egg output of H. polygyrus after 7 days of oral treatment, together with a 79.2% of reduction in total worm counts. Based on the obtained results, L. nobilis EO showed promising in vitro and in vivo anthelmintic capacities against gastrointestinal parasites.

Assessment of anthelmintic potentials of Myrtus communis against Haemonchus contortus and Heligmosomoides polygyrus.

Anthelmintic resistance in grazing livestock systems has been spreading worldwide in prevalence and severity. Therefore, alternative measures including the use of herbal anthelmintic is considered as one of the successful approaches for the control of anthelmintic resistance. In the present report, we describe the chemical constituents of Myrtus communis essential oil, its in vitro anthelmintic effect against the most pathogenic gastrointestinal parasite of sheep; Haemonchus contortus and its in vivo anthelmintic potential using an in vivo gastrointestinal parasite model of rodents; i.e. Heligmosomoides polygyrus. Chromatographic analyzes of the essential oil (EO) extracted from the leaves of M. communis have shown that this oil was composed mainly of a α-pinene (33.59%), eucalyptol (23.85%) and limonene (14.70%). Regarding the in vitro anthelmintic potential, the ovicidal effect was confirmed in an egg hatch inhibition assay at IC50 = 0.7 mg/mL and with 95.83% of immobility of adult worm's after 8 h of exposure to 2 mg/mL of M. communis EO. The anthelmintic capacity of M. communis EO was also confirmed by in vivo assays conducted against the murine parasite H. polygyrus. In fact, at 1200 mg/kg bw of M. communis EO, a reduction of 99.70% in faecal egg counts was observed after 7 days of oral treatment, together with a 71.12% reduction in total worm counts. Based on the obtained results, M. communis EO showed relevant in vitro and in vivo anthelmintic effects against gastro-intestinal parasites.

First insights into the microbiome of Tunisian Hyalomma ticks gained through next-generation sequencing with a special focus on H. scupense.

Ticks are one of the most important vectors of several pathogens affecting humans and animals. In addition to pathogens, ticks carry diverse microbiota of symbiotic and commensal microorganisms. In this study, we have investigated the first Tunisian insight into the microbial composition of the most dominant Hyalomma species infesting Tunisian cattle and explored the relative contribution of tick sex, life stage, and species to the diversity, richness and bacterial species of tick microbiome. In this regard, next generation sequencing for the 16S rRNA (V3-V4 region) of tick bacterial microbiota and metagenomic analysis were established. The analysis of the bacterial diversity reveals that H. marginatum and H. excavatum have greater diversity than H. scupense. Furthermore, microbial diversity and composition vary according to the tick's life stage and sex in the specific case of H. scupense. The endosymbionts Francisella, Midichloria mitochondrii, and Rickettsia were shown to be the most prevalent in Hyalomma spp. Rickettsia, Francisella, Ehrlichia, and Erwinia are the most common zoonotic bacteria found in Hyalomma ticks. Accordingly, Hyalomma ticks could represent potential vectors for these zoonotic bacterial agents.

Zoonotic vector-borne bacteria in wild rodents and associated ectoparasites from Tunisia.

Wild rodents are considered as potential carriers of several zoonotic vector-borne bacteria but their epidemiology is poorly understood in Tunisia. A total of 305 biological samples (100 spleens, 100 livers, 100 kidneys, and 5 pooled ectoparasites (Xenopsylla cheopis, Laelaps echidninus, Ornithonyssus sp., Hoplopleura sp. and eggs of the rat fleas)) were collected from 100 wild rodents from three Tunisian governorates. Molecular screening was performed to reveal infections with main vector-borne bacteria. Captured rodents belonged to three rodent genera and species including Rattus rattus (n = 51, 51%), Meriones shawi (n = 24, 24%) and Mus musculus (n = 25, 25%). Examined rodents were found to be heavily infested by the rat flea X. cheopis (n = 32, 47%) and the rat mite L. echidninus (n = 22, 32.3%). However, the rat mite Ornithonyssus sp. (n = 13, 19.1%) and the rat lice Hoplopleura sp. (n = 1, 1.5%) were rarely identified. Based on 16S rRNA and msp4 genes, infection with Anaplasmataceae bacteria was detected in six specimens of R. rattus and one M. shawi. Pathogenic A. phagocytophilum (n = 1), A. phagocytophilum-like 1 (Anaplasma sp. Japan) (n = 1), and A. ovis (n = 5) were identified. On the basis of ompB, ompA and gltA genes, infection with Rickettsia spp. was identified in three specimens of R. rattus and one of M. shawi. Five Rickettsia species of the spotted fever group, corresponding to R. monacensis, R. helvetica, R. massiliae, R. africae, and R. aeschlimannii, were detected in mixed infections. Bartonella henselae DNA was also found in two R. rattus, based on rpoB partial sequences. All revealed Anaplasma, Rickettsia and Bartonella bacteria were detected in spleen samples. Ehrlichia, Coxiella and Borrelia spp. were not identified in any of the tested samples. In Tunisia, this is the first report indicating infections with Anaplasma, Rickettsia and Bartonella spp. in wild rodents, particularly present alongside domestic livestock and human. This represents a serious risk of potential bacterial transmission. Thus, controlling rodent population in animal herds, residential areas and sensitizing local people to this risk seem absolutely necessary.

Phenology and phylogeny of Hyalomma spp. ticks infesting one-humped camels (Camelus dromedarius) in the Tunisian Saharan bioclimatic zone.

In this study, we report the results of a survey of Hyalomma ticks infesting one-humped camels in southern Tunisia. Examinations were conducted every second or third month on 406 camels in Tataouine district from April 2018 to October 2019. A total of 1902 ticks belonging to the genus Hyalomma were collected. The ticks were identified as adult H. impeltatum (41.1%; n = 782), H. dromedarii (32.9%; n = 626), H. excavatum (25.9%; n = 493), and H. marginatum for a single specimen. Although the camels were infested by ticks throughout the year, the highest overall infestation prevalence was observed in April 2018 (p < 0.01). The overall infestation intensity varied between 2.7 and 7.4 ticks/animal. There were no statistically significant differences in tick infestation prevalence based on age categories of the camels, and the overall infestation prevalence was between 82.7% and 97.4%. Female camels were significantly more infested with ticks (88.3%) than males (65.5%) (p < 0.01). The infestation prevalence of camels varied significantly according to the region where sampling took place (p < 0.01), but no correlations were found with abiotic factors. The preferred attachment sites for adult Hyalomma ticks were the sternum (38.3%; n = 729/1902), around the anus (36.2%; n = 689/1902), udder (18.4%; n = 350/1902), and inner thigh (6.9%; n = 132/1902). Morphological classification of ticks was corroborated by sequencing the cytochrome c oxidase I (Cox1) and 16S rDNA genes, and these sequences were also used to infer phylogenetic relationships. A single H. dromedarii seemed to be a natural hybrid with H. rufipes. More attention should be devoted by the veterinary services to the infestation of camels by ticks.

TITLE: Phénologie et phylogénie des tiques Hyalomma spp. infestant les dromadaires (Camelus dromedarius) dans la zone bioclimatique saharienne tunisienne. ABSTRACT: Dans cette étude, les résultats d'une enquête concernant les tiques Hyalomma infestant les dromadaires dans le sud de la Tunisie sont présentés. Des examens ont été menés tous les deux ou trois mois sur 406 dromadaires dans le district de Tataouine entre avril 2018 et octobre 2019. Au total, 1902 tiques appartenant au genre Hyalomma ont été collectées. Les tiques adultes ont été identifiées comme H. impeltatum (41,1 % ; n = 782), H. dromedarii (32,9 % ; n = 626), H. excavatum (25,9 % ; n = 493) et un seul spécimen de H. marginatum. Bien que les dromadaires aient été infestés par les tiques tout au long de l'année, la prévalence globale d'infestation la plus élevée a été observée en avril 2018 (p < 0,01). L'intensité globale d'infestation variait entre 2,7 et 7,4 tiques/animal. Il n'y avait pas de différence statistiquement significative de l'infestation par les tiques en fonction des catégories d'âge des dromadaires, et la prévalence globale d'infestation se situait entre 82,7 et 97,4 %. Les dromadaires femelles étaient significativement plus infestés par les tiques (88,3 %) que les mâles (65,5 %) (p < 0,01). La prévalence d'infestation variait significativement en fonction des régions (p < 0.01) mais il n'y avait pas de corrélation avec les facteurs abiotiques. Les sites de fixation préférés des tiques Hyalomma adultes étaient le sternum (38,3 % ; n = 729/1902), autour de l'anus (36,2 % ; n = 689/1902), la mamelle (18,4 % ; n = 350/1902) et la face interne de la cuisse (6,9 % ; n = 132/1902). La classification morphologique a été corroborée par le séquençage des gènes de l'ADNr du cytochrome c oxydase I (Cox1) et du 16S, et ces séquences ont également été utilisées pour déduire les relations phylogénétiques. Un seul spécimen de H. dromedarii semblait être un hybride naturel avec H. rufipes. Une plus grande attention doit être accordée par les services vétérinaires quant à l'infestation des dromadaires par les tiques.

First survey of ticks, tick-borne pathogens (Theileria, Babesia, Anaplasma and Ehrlichia) and Trypanosoma evansi in protected areas for threatened wild ruminants in Tunisia.

The aim of the study was to identify ticks present in the environment and wild Tunisian ruminants and to detect tick-borne pathogens and Trypanosoma evansi DNA in these specimens. Sampling was done throughout each season from the environment in three protected areas around Tunisia: El Feidja, Haddaj and Oued Dekouk. Ticks were collected also, from one fawn of Barbary red deer and eight naturally deceased wild ruminants (one Barbary red deer, five Scimitar-horned oryx, one Addax antelope and one Dorcas gazelle), all of which lived in various protected areas. PCR and nested PCRs were performed to detect the presence of Theileria spp., Babesia spp., Trypanosoma evansi, Ehrlichia spp., Anaplasma spp., Anaplasma bovis and Anaplasma phagocytophilum DNA in these tick specimens. A total of 352 ticks were collected, belonging to six different species: Hyalomma excavatum (80.6%), Hyalomma dromedarii (10.2%), Hyalomma marginatum (0.5%), Rhipicephalus bursa (0.5%), Rhipicephalus sanguineus sensu lato (5.1%) and Ixodes ricinus (2.8%). Pathogens have been detected in 25% of H. dromedarii, 9.1% of H. excavatum and 5% of R. sanguineus sensu lato. The percentage of detection of T. evansi was 0.2%. Ehrlichia spp.-Anaplasma spp. were detected in 10.1% of ticks. Anaplasma spp. and A. bovis were detected in 7.6%, and 0.8% of examined ticks, respectively. None of the Theileria spp., Babesia spp., or A. phagocytophilum DNA was detected in the tested ticks. To our knowledge, the present study represents the first identification of these six tick species and the first detection of rickettsial pathogens and T. evansi in North African wild ruminants' species. These results extend the knowledge about the diversity of ticks and tick-borne pathogens in wildlife and justify further investigations of the possible role of R. sanguineus sensu lato in the transmission of T. evansi.

Individual variability among autochthonous sheep in Northern Tunisia to infection by abomasum nematodes and Babesia/Theileria parasites.

In Tunisia, livestock plays an important role in the economy; unfortunately, Tunisian sheep population faces several health challenges. The aim of this trial was to study phenotypic variability of four local sheep breeds and strains to abomasum nematodes infection and to Babesia/Theileria parasites. Faeces, blood and abomasum contents were collected from 310 sheep slaughtered in eight commercial slaughterhouses across North Tunisia. Haematological and biochemical parameters were assessed. DNA was extracted and catch-all primers were used to detect both Theileria spp. and Babesia spp. DNA. Faecal egg counts (FEC) was quantitatively assessed using simple flotation technique followed by McMaster technique. Male and female worms were collected from all abomasum contents and counted under a stereomicroscope. The percentage of faeces samples positive for GIN's eggs was 30.82%. After worms' recovery, the infection prevalence was estimated to 75.90%. The overall infection prevalence by Babesia spp. and Theileria spp. was 4.21%. The dispersion of observations plots obtained by principal component analysis (PCA) showed two clusters of individuals. The first cluster contains animals having positive Babesia/Theileria PCR, presence of nematodes in the abomasum contents and relatively low total worm count (TWC < 500) expect one animal which was found bearing high TWC (>500). In this same group, with a suspected form of resistance, animals showed normal values of albumin and normal haematological parameters (red blood cell count [RBC], haemoglobin [Hb] and packed cell volume [PCV]). The second cluster represents all the other observations in which subgroups of animals were distinguished on the basis of their potential resistance to abomasum nematodes. Multiple correlations showed significant positive correlations between RBC/Hb, RBC/PCV, PCV/Hb and FEC/TWC. Significant negative correlations were observed between TWC/RBC and TWC/Hb. It is concluded that the phenotypic variability among local sheep breeds is essential for more advanced genetic and genomic studies.

Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia.

To date, no information is available regarding the infection of camels (Camelus dromedarius) by Anaplasma ovis in North African region. Several animal species can be infected by A. ovis which further complicates its natural infection cycle. In this paper, we investigated the occurrence and the genetic diversity of A. ovis in camels and ticks collected from them in Tunisia and the risk factor analysis. Camel blood samples (n = 412) and tick (n = 300) samples, identified as Hyalomma dromedarii (n = 149, 49.6%), H. impeltatum (n = 142, 47.3%) and H. excavatum (n = 9, 3%), were analyzed by conventional PCR followed by the sequencing of msp4 and groEL genes. A. ovis DNA was identified in five camels (1.2%), but not in infesting ticks (0%). The microscopic examination revealed the specific infection of camel erythrocytes by Anaplasma inclusions. The msp4 and groEL typing confirmed the natural infection of camels by A. ovis and revealed two different msp4 genotypes earlier detected in Tunisian small ruminants and their infested ticks, and five different and novel groEL genetic variants forming a separately sub-cluster within A. ovis cluster. The occurrence of different A. ovis strains specific to camels associated with a low prevalence of this Anaplasma species in camels may enrich knowledge regarding the distribution and the transmission cycle of this bacterium in arid and Saharan areas of Tunisia.

Improving specific detection and updating phylogenetic data related to Anaplasma platys-like strains infecting camels (Camelus dromedarius) and their ticks.

In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (n = 164, 49%), H. impeltatum (n = 161, 48.3%) and H. excavatum (n = 9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis.

Cystic echinococcosis and other helminth infections of wild boar in northeastern and northwestern regions of Tunisia.

This study identified helminth species of wild boar (Sus scrofa) originating from northeastern and northwestern regions of Tunisia using 297 lungs, 297 livers, 264 intestinal tracts, 120 samples of muscle tissue (tongue, masseter, diaphragm, inter-costal) and 232 faecal samples derived from a total of 591 animals. Host gender was registered for the lung and liver wild boar group, which included 163 males and 134 females. All animals, excluding those used to retrieve muscular samples, were classified into three age classes, <2 (n = 212), 2-3 (n = 208) and ⩾4 years old (n = 141). Helminth fauna of the examined wild boar included 14 parasite species: one trematode (adult, Brachylaemus suis), three cestodes (metacestodes of Echinococcus granulosus, Taenia hydatigena cysticercus, adult, Hymenolepis diminuta), nine nematodes (adults of Metastrongylus apri, Metastrongylus pudendotectus, Ascarops strongylina, Globocephalus urosubulatus, Physocephalus sexalatus, Gnathostoma hispidum, Gongylonema pulchrum and eggs of Strongyloides ransoni and Capillaria spp.) and one acanthocephalan (adult, Macracanthorhynchus hirudinaceus). Trichinella larvae were not recovered from any of the 30 wild boar examined. Results showed a 73.5% global prevalence of infection with visceral helminths, 67.3% of which were lung and hepatic infections and 80.3% of helminths were recovered from the gastrointestinal tract. The most prevalent parasite was M. hirudinaceus (61.7%) while the highest intensity of infection was observed for Metastrongylus spp. The most prevalent cestode was E. granulosus (18.9%). This is the first detailed study on helminth infections of wild boar from a North African country.

Chemical analyses and anthelmintic effects of Artemisia campestris essential oil.

The present study aimed at analyzing the chemical composition and evaluating the in vitro and in vivo anthelmintic activity of Artemisia campestris essential oil aerial parts. The chemical composition was analysed by gaz chromatography/mass chromatography (GC/MS). Fifty compounds were identified representing 99.98% of the total oil. A. campestris essential oil was dominated by beta-pinene (36.40%) and 2-undecanone (14.7%). The in vitro anthelmintic activity tests of A. campestris essential oil were performed on Haemonchus contortus using egg hatch assay (EHA) and adult worm's motility assay (AWMA) compared with a reference drug albendazole. In the EHA 100% inhibition was observed at 2 mg/ml after 48 h incubation (IC50 = 0.93 mg/ml). In the AWMA, essential oil induced 66.6% inhibition at 0.5 mg/ml after 8 h post exposure. The nematicidal effect of essential oil was evaluated on Heligmosomoides polygyrus. It was monitored through faecal egg count reduction (FECR) and total worm count reduction (TWCR). Three doses (2000, 4000 and 5000 mg/kg) were studied using a bioassay. The dose of 5000 mg/kg showed a high nematicidal activity (72.1% FECR and 72% TWCR), 7 days post-treatment. The results of the present study suggest that A. campestris essential oil has a potential anthelmintic activity and further studies are required in order to establish its mechanisms of action.

Reprint of "Echinococcus granulosus sensu stricto (s.s.) from the critically endangered antelope Addax nasomaculatus in Tunisia".

Echinococcus granulosus sensu lato (s.l.) is a zoonotic disease highly endemic in Tunisia. Canids including stray and semi-stray dogs, jackals and foxes are known as definitive hosts and a wide range of ungulates have been shown to harbour the metacestode hydatid stage and may serve as intermediate hosts. Fertile hydatid cysts of Echinococcus equinus and E. granulosus sensu stricto (s.s.) were recently molecularly identified for the first time from Tunisian donkeys. E. granulosus (s.s.) was also identified from wild boars in Tunisia. Here we report the confirmation of hydatid cysts caused by E. granulosus (s.s.) in the critically endangered antelope, Addax nasomaculatus in Tunisia. DNA-based molecular analysis revealed that A. nasomaculatus was infected with E. granulosus (s.s.) which had a 100% identity with the main globally distributed E. granulosus (s.s.) (EgTu01) haplotype. Cysts of Taenia hydatigena (n=33) were also observed on the liver and in the body cavity. Due to their endangered status and their relatively small numbers, it is unlikely that hydatid infection of A. nasomaculatus will form a major contribution to the epidemiology and transmission of E. granulosus in Tunisia, but infection may result in pathology, morbidity and early mortality, and may still play a role in the perpetuation of the parasite in wildlife cycles.

Echinococcus granulosus sensu stricto (s.s.) from the critically endangered antelope Addax nasomaculatus in Tunisia.

Echinococcus granulosus sensu lato (s.l.) is a zoonotic disease highly endemic in Tunisia. Canids including stray and semi-stray dogs, jackals and foxes are known as definitive hosts and a wide range of ungulates have been shown to harbour the metacestode hydatid stage and may serve as intermediate hosts. Fertile hydatid cysts of Echinococcus equinus and E. granulosus sensu stricto (s.s.) were recently molecularly identified for the first time from Tunisian donkeys. E. granulosus (s.s.) was also identified from wild boars in Tunisia. Here we report the confirmation of hydatid cysts caused by E. granulosus (s.s.) in the critically endangered antelope, Addax nasomaculatus in Tunisia. DNA-based molecular analysis revealed that A.nasomaculatus was infected with E. granulosus (s.s.) which had a 100% identity with the main globally distributed E. granulosus (s.s.) (EgTu01) haplotype. Cysts of Taenia hydatigena (n=33) were also observed on the liver and in the body cavity. Due to their endangered status and their relatively small numbers, it is unlikely that hydatid infection of A. nasomaculatus will form a major contribution to the epidemiology and transmission of E. granulosus in Tunisia, but infection may result in pathology, morbidity and early mortality, and may still play a role in the perpetuation of the parasite in wildlife cycles.

Modelling the transmission dynamics of cystic echinococcosis in donkeys of different ages from Tunisia.

During the period from March 2006 to July 2009 a total of 2040 slaughtered donkeys were examined for cystic echinococcosis (CE). CE prevalence in donkeys was 8.48% and the infection pressure (0.0088 infections per year) and infection rate (0.0448 cysts per year) appeared to be lower than those previously reported for cattle, sheep, dromedaries and goats in Tunisia. However, the number of cysts per infection was relatively high (5.07 cysts per infection). Among the 901 collected hydatid cysts the majority were located in the liver (89.9%), 10.09% in the lungs and 4.77% were fertile (43/901). The amplification of a fragment within the mitochondrial gene coding for cytochrome c oxidase subunit 1 (cox1) revealed that donkeys were infected with both Echinococcus equinus (horse strain, G4 genotype) and Echinococcus granulosus (sheep strain, G1 genotype). E. granulosus G1 developed into fertile cysts (15,112 protoscoleces/ml) with a protoscoleces viability of 65.78%. This investigation is the first detailed epidemiological report on cystic echinococcosis infection in donkeys for any endemic region.