First morphometric and molecular characterization of Fasciola spp. in Northwest Tunisia.

The aim of this study was to characterize the Tunisian Fasciola spp. flukes by morphometric and molecular analyses. Flukes were collected from livers of sheep slaughtered in Sejnane slaughterhouses (Bizerte gouvernorate, Northwest Tunisia) between January and March 2021.Five morphometric parameters were determined for all the liver flukes, as follows: (i) total body length (BL), (ii) distance between ventral sucker and the tail (VS-T), (iii) distance between oral sucker and ventral sucker (OS-VS), (iv) abdomen diameter (AD), (v) tail diameter (TD) and the body length to width ratio (BL/BW). Molecular identification of the fluke specimens was carried out by polymerase chain reaction, restriction fragment polymorphism (PCR-RFLP) of a 680 bp sequence of the internal transcribes spacer 1 (ITS1) gene and by amplification, sequencing, and phylogenetic analysis of a 500 bp sequence of the ITS2 gene. Morphometric measurements showed that the mean of the total body length of the adult flukes was 21.1 ± 2.7 mm with minimum and maximum lengths of 13 and 31 mm, respectively. The PCR-RFLP analysis revealed a single profile consisting of three bands of approximately 370, 100, and 60 bp. Fasciola sequences described in the present study (GenBank numbers: OQ457027 and OQ457028) showed 99.58-100% identity to Fasciola hepatica. In conclusion, the results of this study show that molecular and phylogenetic analyses confirm the presence of a single species of F. hepatica in the Sejnane region Northwest of Tunisia. However, further studies are needed to identify the occurrence of Fasciola species in other Tunisian regions.

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.

Isolation, Identification, and Characterization of Phosphate-Solubilizing Bacteria from Tunisian Soils.

Soil microorganisms play an important role in maintaining natural ecological balance through active participation in carbon, nitrogen, sulfur, and phosphorous cycles. Phosphate-solubilizing bacteria (PSB) are of high importance in the rhizosphere, enhancing the solubilization of inorganic phosphorus complexes into soluble forms available for plant nutrition. The investigation of this species of bacteria is of major interest in agriculture, as they can be used as biofertilizers for crops. In the present study, 28 isolates of PSB were obtained after the phosphate enrichment of soil samples from five Tunisian regions. Five PSB species were identified by 16S rRNA gene sequencing including Pseudomonas fluorescens, P. putida, and P. taiwanensis, Stenotrophomonas maltophilia, and Pantoea agglomerans. Solid and liquid Pikovskaya's (PVK) and National Botanical Research Institute's (NBRIP) media containing insoluble tricalcium phosphate were used for the evaluation of the phosphate solubilization ability of the bacterial isolates by two methods: visual evaluation of the solubilization zone around colonies (halo) and determination of solubilized phosphates in liquid medium by the colorimetric method of the vanado-molybdate yellow. Based on the results of the halo method, the isolate of each species that showed the higher phosphate solubilization index was selected for evaluation of phosphate solubilization by the colorimetric method. In the liquid media, the bacterial isolates showed phosphate solubilization ranging from 535.70 to 618.57 µg mL-1 in the NBRIP medium, and 374.20 to 544.28 µg mL-1 in the PVK medium, with the highest values produced by P. fluorescens. The best phosphate solubilization ability and higher reduction in broth pH, which indicates higher organic acid production, were achieved in NBRIP broth for most of the PSB. Strong correlations were observed between the average capability of PSB to solubilize phosphates and both the pH and total phosphorous content in the soil. The production of the hormone indole acetic acid (IAA), which can promote plant growth, was observed for all five PSB species. Among them, P. fluorescens obtained from the forest soil of northern Tunisia showed the highest production of IAA (50.4 ± 0.9 µg mL-1).

Detection and molecular identification of Anaplasma phagocytophilum and Babesia spp. infections in Hyalomma aegyptium ticks in Tunisia.

Tortoises of the genus Testudo are the main hosts of Hyalomma aegyptium ticks. This species serves as a vector of several zoonotic pathogens. Therefore, the present study aimed to investigate the presence of four pathogens associated with H. aegyptium ticks obtained from tortoises from Tunisia. Conventional, multiplex and nested PCRs were used for Aanaplasma phagocytophilum, Ehrlichia canis, Coxiella burnetii and Babesia spp. screening. The molecular analyses revealed the presence of A. phagocytophilum and Babesia spp. None of the ticks, were infected by E. canis or C. burnetii species. Co-infection was detected in four ticks. As a conclusion, this is the first detection of A. phagocytophilum and Babesia spp. in H. aegyptium ticks collected from tortoises, in Tunisia. Thus, considering these results, the spur-thighed tortoise constitute a potential host of H. aegyptium which plays an important role in the transmission of pathogenic agents affecting both human and animals. In term of public health, a strict control and surveillance should be carried to reduce the circulation of such pathogens between different hosts.

Salmonella Broiler Meat's Contamination in Tunisia: Prevalence, Serotypes, Antimicrobial Resistance and Molecular Characterization of Isolated Strains.

This study was conducted in north-eastern Tunisia to estimate the contamination prevalence of Salmonella in broilers' meat, to rank serotypes and to characterize the isolated multidrug-resistant (MDR) strains. A total number of 1288 meat samples were collected from 322 broiler batches; Salmonella isolates were identified by the alternative technique VIDAS Easy Salmonella. The susceptibility of Salmonella isolates was assessed against 21 antimicrobials using the disc diffusion method on Mueller-Hinton agar. Some antimicrobial resistance genes were identified using Polymerase Chain Reaction (PCR). The prevalence rates of Salmonella in the neck skin and the breast muscle contamination were estimated at 11.8% (38/322) and 0.9% (3/322), respectively. The prevalence rate of Salmonella in meat cutting parts contamination was estimated at 5.1% (33/644). Eight serotypes of Salmonella were identified, namely S. Enteritidis, S. Kentucky, S. Anatum, S. Infantis, S. Mbandaka, S. Zanzibar, S. Hadar and S. Agona. High rate of resistance was identified against amoxicillin (91.9%), nalidixic acid (83.8%), tetracycline (75.7%), streptomycin (73%), ciprofloxacin (70%), sulfamides (68.9%), cefalotin (68.9%), cefotaxim (67.6%) and cefoxitin (60.8%). The majority (90.5%; 67/74) of isolated strains was recognized as MDR. Nine MDR strains were identified as Extended-Spectrum ß-Lactamase (ESBL) producers. The blaCTX-M gene was identified by PCR in all the nine ESBL strains. TetA, tetB and dfrA1 genes were amplified in 3.6% (2/56), 1.8% (1/56) and 19.3% (5/26) of tetracycline and trimethoprim-resistant strains, respectively. The integrase gene (class 2) was identified in only 8.1% (6/74) of the Salmonella-isolated strains. Our findings highlight the emergence of MDR Salmonella isolates in Tunisia.

Molecular identification of Neospora caninum and co-infection with Toxoplasma gondii in genital apparatus of naturally infected cows in North Tunisia.

The aim of this study was to estimate the molecular prevalence of Toxoplasma gondii, Neospora caninum and the co-infection by both Apicomplexan parasites in uterus tissues of cows. PCR was used to detect T. gondii and N. caninum DNA in uterus from 140 uteri of slaughtered cows in the regional slaughterhouse of Béja (Northwest Tunisia). Positive PCR products were sequenced and used for the phylogenetic analysis. The overall molecular prevalence of T. gondii and N. caninum in cows' uterus was 5 and 15.57%, respectively. Co-infection prevalence by the two parasites was estimated to be 2.85%. Risk factors including the age categories significantly affected the molecular prevalence of T. gondii and N. caninum in cows' uterus. The highest molecular prevalence of T. gondii (11.5 ± 3.1) and N. caninum (21.1 ± 11.1; p = 0.038) was observed in cows aged of more than 8 years. There were no differences depicted according to cow's breeds and localities. Comparison of the partial sequences of the ITS1 gene revealed 100% similarity among our N. caninum sequence (MW136256) and those deposited in GenBank. The T. gondii sequence described in this study (MW260335) was 99.4-100% homologous to T. gondii sequences published in the GenBank.To the best of our knowledge, this is the first molecular evidence of N. caninum and T. gondii co-infection in naturally infected cows in North Africa. This information is pertinent in designing control programmes that would reduce economic losses in the livestock industry.

Prevalence, Risk Factors, Antimicrobial Resistance and Molecular Characterization of Salmonella in Northeast Tunisia Broiler Flocks.

This study was conducted in northeastern Tunisia to estimate both the prevalence and the risk factors of Salmonella in broiler flocks as well as to characterize the isolated multidrug-resistant (MDR) Salmonella strains. In the present study, a total number of 124 farms were sampled; Salmonella isolates were identified by the alternative technique VIDAS Easy Salmonella. The susceptibility of Salmonella isolates was assessed against 21 antimicrobials using the disk diffusion method on Mueller-Hinton agar using antimicrobial discs. Some antimicrobial resistance genes were identified using PCR. The prevalence rate of Salmonella infection, in the sampled farms, was estimated at 19.9% (64/322). Moreover, a total number of 13 different serotypes were identified. High rate of resistance was identified against nalidixic acid (82.85%), amoxicillin (81.25%), streptomycin (75%), and ciprofloxacin (75%). Alarming level of resistance to ertapenem (12.5%) was noticed. A total of 87.5% (56/64) of isolated strains were recognized as MDR. Three MDR strains were extended-spectrum ß-lactamases (ESBL)-producers and three MDR strains were cephalosporinase-producers. The blaCTX-M gene was amplified in all the three ESBL strains. The qnrB gene was not amplified in fluoroquinolones-resistant strains. The tetA and tetB genes were amplified in 5% (2/40) and 2.5% (1/40) of tetracycline-resistant strains, respectively. The dfrA1 gene was amplified in five of the 20 trimethoprim-resistant strains. The mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 genes were not amplified in any of the phenotypically colistin-resistant strains. In terms of integrase genes int1 and int2, only gene class 2 was amplified in 11% (7/64) of analyzed strains. Risk factors, such as the poor level of cleaning and disinfection, the lack of antimicrobial treatment at the start of the breeding, and a crawl space duration lower than 15 days, were associated with high Salmonella infection in birds. These data should be considered when preparing salmonellosis control programs in Tunisian broiler flocks.

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.

Blood Meal Analysis of Phlebotomine Sandflies (Diptera: Psychodidae: Phlebotominae) for Leishmania spp. Identification and Vertebrate Blood Origin, Central Tunisia, 2015-2016.

During the time periods of June 2015 and from July to August 2016, sandflies were collected among seven collection sites of the three leishmaniasis endemic villages of Sidi Bouzid, Tunisia. A total of 690 sandflies were captured and identified (380 males and 310 females). Four species belonging to genus Phlebotomus (Ph.) and two species belonging to genus Sergentomyia were identified. Leishmania DNA was detected in four out of 310 females (one Ph. sergenti and three Ph. papatasi). The overall sensitivity of the Prepronociceptin gene detection reached 76%. The concurrent presence of Ph. papatasi and Ph. sergenti vectors, the analysis of blood-meals, together with the detection of L. major in Ph. papatasi, confirms the ultimate conditions for the transmission of the disease in center Tunisia. These results expand the known epidemiological area of distrubtion of leishmaniasis and its vectors in this part of Tunisia, highlighting the need for ongoing entomological and parasitological surveillance.

First Report on Natural Infection of Phlebotomus sergenti with Leishmania tropica in a Classical Focus of Leishmania major in Tunisia.

In Tunisia, chronic cutaneous leishmaniasis due to Leishmania tropica is an important health problem. Its spreading has not been fully elucidated. Information on sandfly vectors, as well as their associated Leishmania species, is of paramount importance since vector dispersion is one of the major factors responsible for pathogen dissemination. Ninety-seven unfed females belonging to the genera Sergentomyia and Phlebotomus were collected between June and August 2015 using sticky paper traps. Polymerase chain reaction-restriction fragment length polymorphism analysis of the internal transcribed spacer 1and sequencing were used for Leishmania detection and identification. In total, 650 sandflies were captured and identified (380 males and 270 females). Ninety-seven unfed females were tested for the presence of Leishmania parasite DNA. Six Phlebotomus sergenti were found positive for L. tropica. This novel finding enhances the understanding of the cycle extension of L. tropica outside its original focus of Tataouine.

Molecular detection of Toxoplasma gondii infection in slaughtered ruminants (sheep, goats and cattle) in Northwest Tunisia.

The present study aimed to estimate the molecular prevalence of T. gondii infection in meat from slaughtered sheep, goats and cattle in Northwest Tunisia (Béja district). PCRs were performed on genomic DNA extracted from 420 meat samples (150 ewes, 120 goats and 150 cows). The overall molecular prevalence of T. gondii in sheep, goats and cattle were 33.3 (50/150), 32.5 (39/120) and 19.3% (29/150), respectively. Toxoplasma gondii molecular prevalences in the three meat ruminant species were significantly higher in adults compared to young animals (p<0.001). The infection prevalence differed significantly within localities in sheep (p<0.001), goats (p<0.001) and cattle (p=0.019). These results provide important information about exposure of humans to T. gondii through the consumption of raw or undercooked ruminants' meat. Extension programmes should be implemented to decrease the risk of infection related to sheep, goats and cattle meat manipulation and raw or undercooked meat consumption.

First molecular detection and characterization of Sarcocystis species in slaughtered cattle in North-West Tunisia.

Sarcocystis spp. is one of the most prevalent foodborne parasites infecting both animals and humans. Consumption of raw or undercooked infected meat is a risk factor of human intestinal sarcocystosis. The present study aimed to estimate the prevalence of Sarcocystis species infecting slaughtered Tunisian cattle in North-West Tunisia (Béja governorate). DNA was extracted from 150 beef meat samples and a PCR-restriction fragment length polymorphism was used for identification. The overall infection prevalence of Sarcocystis spp. was 38% (57/150). Two species were identified, namely S. hominis (25%; 39/150) and S. cruzi (12%; 18/150). For both species, the highest prevalence was in Thibar locality (52.9 and 17.6% for S. hominis and S. cruzi, respectively). The molecular prevalence of S. cruzi was significantly higher in animals aged between two and eight years (19.2%; 10/52). This is the first molecular identification of Sarcocystis species in Tunisian cattle. Further studies in both human and animal Tunisian populations are needed to rank this parasitic disease among others.

Prevalence, virulence and antibiotic susceptibility of Salmonella spp. strains, isolated from beef in Greater Tunis (Tunisia).

The aim of this work was to investigate the presence of Salmonella spp. in 300 beef meat samples collected from cattle carcasses of different categories (young bulls, culled heifers and culled cows). The detection of Salmonella spp. was performed by the alternative VIDAS Easy Salmonella technique and confirmed by PCR using Salmonella specific primers. Salmonella serotypes were determined by slide agglutination tests. The resistance to 12 antibiotics was determined by the diffusion method on Mueller-Hinton agar antibiotic discs. The overall contamination rate of beef by Salmonella spp. was 5.7% (17/300). This rate varied from naught (0/100) in bulls' meat to 14% (14/100) in culled cows' meat (p<0.001). The prevalence of Salmonella spp. was higher in summer and in cattle with digestive disorders: chronic gastroenteritis (6/17), traumatic peritonitis (3/17) and intestinal obstruction (2/17) (p<0.0001). Of the 17 Salmonella isolates, 6 serotypes were identified, namely Salmonella Montevideo (8/17), Salmonella Anatum (3/17), Salmonella Minnesota (2/17), Salmonella Amsterdam (2/17), Salmonella Kentucky (1/17) and Salmonella Brandenburg (1/17) (p<0.05). Unlike other serotypes, S. Montevideo was present during the whole year except winter. Almost all of the strains (16/17) were resistant to at least one of the 12 tested antibiotics. Multidrug-resistance concerned 14/17 of the strains, including Amoxicillin (13/17), Tetracycline (12/17), Streptomycin (10/17) and Nalidixic acid (6/17). All the strains were sensitive to the association (Amoxicillin+Clavulanic acid), Cefoxitin and Ceftazidime. In addition, our study showed that all Salmonella strains (17) were positive for invasion gene invA and negative for the virulence gene spvC. Only one isolate (S. Kentucky) harbored the h-li virulence gene.

Infestation of the spur-thighed tortoise (Testudo graeca) by Hyalomma aegyptium in Tunisia.

We examined 210 spur-thighed tortoises (Testudo graeca) for the presence of ticks in Tunisia during May 2014. A total number of 602 adult ticks were collected and identified leading to the estimation of parasitological indicators. All the ticks belonged to a single species: Hyalomma aegyptium. The mean infestation prevalence was 66.2%, mean overall infestation intensity and abundance were 4.33 and 2.86 ticks/tortoise respectively. Our survey showed that tortoises were significantly more infested by male ticks than females (p<0.001). The ticks were mainly present in the posterior limbs compared to other body regions (p<0.05). There was no significance variation of length and weight of tortoises according to sex (p<0.05). There was a significant correlation between the tortoises' size (length and weight) and tick infestation. This study showed high tick burdens of spur-thighed tortoises in Tunisia; further investigations are needed to determine exactly the role of this tick species in the transmission of different zoonotic pathogens.

First report of surra (Trypanosoma evansi infection) in a Tunisian dog.

Trypanosoma evansi, the agent of surra, is a salivarian trypanosome, originating from Africa. Surra is a major disease in camels, equines and dogs, in which it can often be fatal in the absence of treatment. Animals exhibit nonspecific clinical signs (anaemia, loss of weight and abortion). In the present survey, a blood sample was collected in Sousse (Central Tunisia) from a dog that presented clinical signs of trypanosomiasis. Giemsa-stained blood smears and PCR were performed. ITS1 sequences from blood had 99.8 and 99.5% homology with published T. evansi sequences from cattle and camels, respectively. To our knowledge, this is the first report of T. evansi in a Tunisian dog.

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.