Ethics statement
All animal experiments were performed according to the European Convention on the protection of animals used for scientific purposes (EU Directive 2010/63/EU) and approved by the local Experimental Animals Ethics Committee of the Faculty of Pharmacy, University of Monastir, Tunisia. Approval for the use of animals and all procedures was obtained from the Ethics Committee of the Faculty of Medicine, University of Monastir under ethics number IORG 0009738N°21/ OMB 0990 − 0279. During the study, all methods were carried out in accordance with relevant guidelines and regulations and efforts were dedicated to minimize the suffering of used animals.
For butchers and chicken owners, the study was conducted in accordance with relevant guidelines and regulations and after ethical clearance obtained from the Ethics Committee of the Faculty of medicine, University of Monastir Tunisia (Code IORG 0009738N°21/ OMB 0990 − 0279). Verbal informed consent was obtained and recorded from each participant after adequate explanation of the study purpose. Only butchers and chicken owners who agreed to participate were interviewed.
Study Area And Sample Collection
This study is an analytical cross-sectional study. It was conducted in two different areas in Tunisia: the first is located in Central-Eastern Tunisia in the coastal city of Monastir; while the second is located in the South-West of the country in Gafsa.
The study was carried out in compliance with the “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guidelines version 2.0. Thus, between September 2016 and May 2018, samples of blood, heart and / or brain were randomly collected from a total of 766 domestic animals reared in the region of Monastir and Gafsa. Only, animals aged of more than three months were included in this investigation.
A structured questionnaire which included the sex, age, specie, breed, origin and living area of the animal, was used.
Blood and heart tissue samples were collected from 630 sheep (Ovis aries) slaughtered in slaughterhouses from two regions: Monastir and Gafsa (315 from each). Two breeds represented these animals: the Barbarine breed (n = 183) and the Queue fine de l’Ouest breed (n = 447).
In addition, samples of blood, brain and heart tissue were taken from each of the 136 free-range chickens (Gallus gallus domesticus) collected from farms and backyards (76 from Monastir and 60 from Gafsa).
Chicken blood was withdrawn from the wing vein using an insulin syringe (1 mL). These chickens were then marked with adhesive tape. The purchase of the animal and the removal of the heart and brain were done after a positive serology test. Sampling was conducted both in urban and rural localities in each region.
After centrifugation 10 min at 3000 rpm, sera were stored at -20 °C until use. The hearts were removed sterilely, with a single-use scalpel blade, entirely for chickens and partly for sheep (apex of cardiac muscles) and the brains of the chickens were removed entirely by opening the cranial box. The tissue samples were placed in mixed saline solution of antibiotics with (1000 U / mL of penicillin and 10 mg / mL of streptomycin) and stored at + 4 °C before processing (Table S1).
Serological Examination
Sera were tested for T. gondii specific IgG antibodies in chicken and sheep using the highly sensitive direct agglutination test (DAT) (Toxo-Screen DA, bioMérieux®, France) according to manufacturer instructions; sera was diluted to 1:40, 1:60, 1:180, 1:540, 1:1620 and 1:4000 with a seropositivity cut-off at 1:40 dilution titer.
Bioassay of tissue samples for T. gondii
The isolation protocol was carried out as indicated previously with some modifications 25.
Brain and cardiac muscle tissues from seropositive domestic animals (≤ 50 g) were blended, homogenized in saline solution (0.9% NaCl) with a trypsin solution (0.25%) (Eurobio, Courtaboeuf, France) incubated in a shaker water bath at 37 °C for 90 min. The suspension was then filtered through two layers of gauze, the pellet was washed three times by centrifugation for 10 min at 2600 rpm, then resuspended in 0.9% NaCl, 200 µL of the suspension were stored at -80 °C until DNA extraction. Finally, the digestate pellets were incubated with antibiotics (1000 U/mL penicillin and 10 mg/mL streptomycin) at 4 °C overnight before inoculation.
About 1 mL of homogenate was inoculated intraperitoneally into each of four mice (female Swiss Webster mouse 20–25 g). Mice were monitored daily for clinical signs of toxoplasmosis.
Four weeks after inoculation, blood collection from mice was performed from the retro-orbital sinus of the eye for serological screening. The antibodies directed against T. gondii were determined by the DAT test (Toxo-Screen DA, bioMérieux®, France).
Six weeks after inoculation, the brains of seropositive mice were removed aseptically. All brains and positive digestates were transported to T. gondii Biological Resource Center (BRC Toxoplasma) of Limoges, for genotyping studies.
The brain was homogenized with 1 mL 0.9% NaCl using a 5 mL and 2 mL syringues with a 23 and 20 G needles respectively. After microscopic examination, 200 µL of each brain tissue were stored at -20 °C until DNA extraction.
Strains were cryopreserved in nitrogen liquid with RPMI containing 10% FCS and 10% DMSO at BRC Toxoplasma, Limoges, France (http://www.toxocrb.com).
DNA extraction and genotyping of T. gondii isolates
Genomic DNA was extracted from 200 µL of mouse brain tissue for each strains and directly from the digestate of animal seropositive tissue, using the QIAamp DNA MiniKit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations.
We firstly checked the presence of toxoplasmic DNA in the digestate of seropositive animals by conventional PCR as mentioned in 32.
Secondly, before genotyping we estimate the amount of Toxoplasma DNA from digestates of PCR positive animal tissues for which we failed to isolate the strain using a real-time quantitative PCR (qPCR) targeting the 529 bp repeat region of T. gondii DNA fragment (GenBank accession number AF146527) 40, as described previously 41.
All brains from seropositive mice and animal tissue digestates with a Cq value less than 32 by qPCR were subjected to genotyping analysis using 15 microsatellite markers, as previously described 16. These markers included 8 typing markers (TUB2, W35, TgM-A, B18, B17, M33, IV.1, XI.1), showing little or no variation within lineages and 7 “fingerprinting” markers (M48, M102, N83, N82, AA, N61, N60) showing significant polymorphism variation within lineages.
Genetic Clustering
In order to quantify the extent of genetic distance between Tunisian populations and evaluate their position in relation to the previously described genetic diversity of T. gondii strains, a Neighbour-joining (NJ) tree was constructed from the genetic distances among individual isolates using Populations 1.2.32 (http://bioinformatics.org/populations/) based on Cavalli-Sforza and Edwards chord distance estimator 42 and generated with MEGA 6.05 (http://www.megasoftware.net/history.php).
Reference strains representing the 16 T. gondii haplogroups (HGs) described to date 17,43
were used for comparison with strains from this study: GT1 (HG1), ME49 (HG2), VEG (HG3), MAS (HG4), RUB (HG5), FOU (HG6), CAST (HG7), TgCtBr5 (HG8), P89 (HG9), VAND (HG10), COUG (HG11), ARI (HG12), TgCtPRC04 (HG13), TgA105004 (HG14), TgCtCo5 (HG15) and CASTELLS (HG16). TgCatEg65 4,44 was used as the reference strain for Africa 4 lineage. In addition, human strains previously isolated from cases of congenital toxoplasmosis in Tunisia 32 were also included. Finally, a number of animal isolates from Algeria 45, Ethiopia 46, and Gabon 25 were included in order to compare Tunisian strains to other African strains.
A Minimum spanning network (MSN) was generated usin “Poppr” package 47 (implemented in R environment) to evaluate the geographical segregation between the genotypes from Monastir and Gafsa. HP-Rare 1.1.48 was used to compare allelic richness between the two regions using a rarefaction procedure.
A second MSN was generated in order to evaluate the extent of migrations of T. gondii strains between Tunisia and other regions of the world. The MSN included genotypes from this study and a set of previously published genotypes mainly originating from mediterranean countries (Algeria, Egypt, France, Turkey) and Austria.
Discriminant analysis of principal components (DAPC), implemented in the ADEGENET package in the R environment 49, was performed to infer population subdivision within the same set of genotypes from Tunisia and other countries. This nonparametric approach (free from Hardy–Weinberg assumptions) makes no assumptions regarding data structure or underlying population genetics model, and is therefore suitable for organisms which display high levels of clonality such as T. gondii. In this model, genetic data is initially transformed using a principal components analysis (PCA), followed by a discriminant analysis (DA) to identify clusters. The optimal number of clusters (populations) is calculated using the k-means clustering algorithm, based on the Bayesian information criterion (BIC), which reaches its minimum when approaching the best supported assignment of individuals to the appropriate number of clusters. Individuals having less than 90% of probability of membership in a single cluster were considered as admixed 50. R packages were run in R sofware version 3.4.0.