DOI: https://doi.org/10.21203/rs.3.rs-127138/v1
The isolation and molecular typing of Toxoplasma gondii strains provides an essential basis for a better understanding of the distribution of genetic diversity and associated human health risks. In this context, we report, the molecular characterization of strains of T. gondii isolated from domestic animals infected with T. gondii in Southern and coastal area of Tunisia. This diversity is compared to that known for the Western European and Mediterranean sub-region.
Blood, hearts and/or brains were collected from 766 domestic animals (630 sheep and 136 free-range chickens). Strain isolation from these samples were performed using mouse bioassay. The strains genotyping was carried out with a multiplex PCR technique using 15 microsatellite markers.
Viable strains of T. gondii were isolated from 13.4% sheep and 33.3% chickens. Furthermore, the parasite was also detected in three DNA extracts from animal tissue digestates.
This study showed a large predominance of type II strains (87.9%) from which two samples were type II variants for W35 locus. The other genotypes were three type III sheep isolates (9.1%) and, for the first time in Tunisia, an isolate of sheep origin of the Africa 4 genotype (3.0%). The comparison of microsatellite alleles of type II strains shows the recent migration of strains between Tunisia and other countries of the world.
Toxoplasma gondii is a zoonotic apicomplexan parasite infecting humans and other warm-blooded animals including livestock 1,2 and poultry 3,4. The latters serve as an intermediate host, while felines are the final hosts of T. gondii 5. It is estimated that T. gondii infects one third of the world's human population 6. Humans are infected by ingesting tissue cysts from undercooked or raw meat or by accidental ingestion of oocysts released into the environment in feline feces 7,8.
Toxoplasmosis is usually asymptomatic in healthy individuals. However, it can be serious or even fatal in the immunocompromised person 9–11 or after congenital transmission 12,13. It also has a significant economic impact, leading to numerous abortions in small farmed ruminants14.
First genotyping studies have suggested the existence of clonal populations for Toxoplasma with three main lineages: types I, II and III and rare recombinants strains 15. In recent years, greater genetic variability has been demonstrated using multilocus markers. Thus, a large number of studies using these genotyping techniques have increased our knowledge of the distribution and global diversity of T. gondii 16–19 with the identification of varying strain populations in different regions of the world. The worldwide distribution of T. gondii genotypes is well-known in Europe 20,21, but yet less explored in Asia and Africa 22–24. In Africa beside the intercontinental lineages type II and type III, African lineages named Africa 1, 2, 3 and recently Africa 4 have been identified using microsatellite markers (MS) or PCR-restriction fragment length polymorphism (RFLP) 4,24−28. Other lineages or strain genotypes were rarely isolated, and the diversity of strains in the wild is still unknown 24.
In Tunisia, previous studies mention a high prevalence of T. gondii in humans and domestic animals, but the genetic diversity of strains circulating in the country is still poorly known. One study performed on sheep meat from Tunis area, Northern Tunisia, shows the existence of types I, II and III and type II/III strains 29. Two genotyping studies were carried out on congenital cases of toxoplasmosis in the same region: the first study is a case of death associated with a recombinant genotype I/III 30. In the second study, the genotypic analysis of the isolates from 14 cases of congenital toxoplasmosis by multilocus RFLP revealed the presence of different genotypes including type I, admixed genotypes with type I and type III alleles (I/III) or type I and type II alleles (I/II) and genotypes with multiallelic profiles for some markers (two to three alleles for the same locus) 31.
However, the above-mentioned studies have been controversial as this genotyping diversity was contrasting with the one of other North African countries, in which type II was the predominant lineage and in which type I strains have never been isolated. In addition, the uncommon proportions of mixed infections with abundance of type I alleles, raised concerns about a possible DNA contamination of samples with RH strain (a type I strain usually used in many laboratories as a PCR positive control), favored by the use of nested PCR techniques.
In a recent study, the strains involved in 4 cases of human congenital toxoplasmosis in Monastir area 32 were isolated by mouse bioassay and genotyped using a highly discriminatory method based on the analysis of 15 microsatellite markers 16. The four strains were found to be of type II lineage, a results more in tune with the diversity of strains expected to be found in a North African country 24.
In view of these discordant results, it is therefore interesting to isolate strains from the domestic fauna in Tunisia and to genotype these strains using highly discriminatory markers16, in order to determine which strains are truly circulating in this country. Notably, these markers have enough resolutive power to discriminate between different strains of the same lineage, enabling to distinguish type I laboratory strain (RH) from natural type I strains and therefore to detect DNA contamination issue.
In this context, the objective of this study was to describe the diversity of T. gondii strains circulating in Tunisia among domestic animals intended for human consumption. The sampling efforts were focused on two regions of the country: the coastal city of Monastir and the inland region of Gafsa. In order to estimate the extent of strain migrations between T. gondii populations from Tunisia and other regions of the world, we compared the genotypes of this study with those of previous studies, focusing mainly on other Mediterranean countries.
The high sensitivity direct agglutination technique detected IgG antibodies against T. gondii in 175 (22.8%, 95% confidence interval CI 19.87–25.82) of 766 sera of animals. Seroprevalence was 22.5% [95% CI 19.24–25.76] and 24.3% [95% CI 17.09–31.51] in sheep and chickens, respectively.
Isolated T. gondii strains
Viable strains of T. gondii were isolated from 13.4% (19/142) sheep and 33.3% (11/33) chickens. All the isolated strains were non virulent for mice.
Sheep isolates were designated as TUN-Ovi ari-62 to 78 and chickens’ isolates were designated by TUN-Gal dom-34 to 42, 48 and 52 (Table S1). Toxoplasma PCR was positive in 100 animal tissue digestates, 30 of which resulted in isolation of the strain after mice inoculation. Of the other 70 digestates tested with qPCR, only 5 samples presented Cq values < 32 (from 25.4 to 31) which is the threshold to consider microsatellite genotyping.
Genotyping was performed on all live strains (30 strains) and on qPCR positive samples with Cq˂ 32 (5 DNA isolates for which the strain could not be genotyped on the mouse brains) (Table S2). Of 35 positive DNA extracts, 33 (21 sheep and 12 chickens) were successfully genotyped (94.3%). Thirty-two strains were fully genotyped by microsatellite analysis (15/15 MS markers) and one with 14/15 MS markers. Most of the T. gondii isolates (29/33; 87.9%) belonged to type II lineage. This allowed identifying in Monastir region, 13 type II isolates (10 sheep / 3 chickens), three isolates of sheep type III, and, for the first time in Tunisia, an Africa 4 genotype in one ovine isolate. In the region of Gafsa, there were seven sheep isolates of type II and nine chicken isolates of type II of which two were single repeat variants at the W35 locus (allele 244 bp instead of 242 bp) of the ME49 type II reference strain (Table S2). Microsatellite analysis of isolates revealed no mixed infection in both studied regions.
In the NJ tree (Fig. 1), most Tunisian genotypes clustered into one of three groups corresponding to three clonal lineages: type II, type III and Africa 4. The type III strain TUN-Oviari-069 showed some degree of divergence from the type III cluster in the tree, likely due to an unusual allele at the M102 marker (176 instead of 190). Type II genotypes showed several subdivisions within the type II cluster in the NJ tree. Most type II genotypes from Tunisia (24/29) segregated from type II genotypes from other countries (Algeria, Ethiopia and USA) and gathered in three branches nearly exclusively composed of Tunisian genotypes. These branches included the Tunisian genotypes from human isolates 32 from the same area. Other branches included type II genotypes from Tunisia (5/29) and other countries.
The paucity of strains belonging to type III and Africa 4 lineages precluded performing extensive analysis of these lineages and hence the following analyses focused on strains of type II lineage.
The MSN including type II genotypes from Monastir and Gafsa showed no geographical structure between the T. gondii populations from two Tunisian regions (Fig. 2a). Monastir and Gafsa had highly similar values of mean allelic richness, of 2.93 and 2.90, respectively.
The MSN including type II genotypes from Tunisia and 5 other countries (Algeria, Egypt, France, Turkey and Austria) showed that the majority of Tunisian genotypes cluster together and segregate from other geographical populations, with few exceptions (Fig. 2b). Notably, 2 of the 5 genotypes from Algeria included in this MSN clustered with the Tunisian group.
Using a selection model based on Bayesian information criterion (BIC) values, the optimal number of genetic clusters was K = 5 among type II genotypes from the six countries (Fig. 3a, b). The genetic cluster 5 was predominant among Tunisian genotypes (9/22) followed by cluster 3 (4/22), in addition to 9 admixed genotypes. Genotypes of Cluster 5 or with admixed profiles which cluster 5 were uncommon outside Tunisia and were found in Algeria (2/5), France (2/35) and Turkey (1/21) (Fig. 3c).
In this study, the main aim was the isolation and genotyping of viable T. gondii from sheep and free-range chickens in Tunisia destined for human consumption. Thirty strains were isolated and three DNA extracted directly from the tryptic digestion pellets for inoculation, which did not allow the isolation of strains. A total of 33 of these T. gondii strains and DNA
isolates from free-ranging chickens and sheep were successfully genotyped (Table S2). Most of the T. gondii strains circulating in the study areas belonged to the type II clonal lineage. For the region of Monastir, in addition to type II strains, three type III strains and, for the first time in Tunisia, an Africa 4 strain. This apparently higher diversity of lineages in Monastir compared to Gafsa can be attributed to the fact that the sheep slaughtered in this region often come from different governorates of the country. At the opposite, the region of Gafsa is a center for breeding livestock, so all slaughtered sheep are native to this region, which may explains the limited diversity of lineages observed in the samples from this region. However, these observations were made on a relatively small sampling, and should be confirmed by larger scale studies.
In addition, we found no difference of allelic richness between the two regions when considering type II lineage only, suggesting that the diversity of strains from Monastir is not obviously higher than that from Gafsa for this lineage.
The preponderance of the clonal type II lineage in this study is consistent with the results of previous studies that showed more generally the predominance of this lineage in North Africa 24 and in other countries of the Mediterranean basin 33–36. The dissemination of this lineage in these regions could be explained by the privileged trade exchange through both maritime and terrestrial routes for millennia, through the circulation of infected animals (cats, rodents and livestock herds) 23,24.
The predominance of type II lineage is also in accordance with our data on human cases of congenital toxoplasmosis in Monastir area, which also revealed the presence of type II 32. Type II genotypes were also found in poultry from Tunis, Northern Tunisia 37.
The comparison of the type II genotypes from Monastir and Gafsa using a Minimum Spanning Network showed no geographical structure between the T. gondii populations from two regions, indicating that strains of this lineage circulate within the country. Indeed, the sheep breeding centers connect the inland regions to the coast and the North regions of Tunisia by commercial movements of sheep herds. This connection is more accentuated during the sacrifice feast (Aïd Al-Adha) when an infected sheep from one region could be slaughtered in another and its offal could be consumed by cats leading to a regional dissemination of this lineage 25.
Population genetics analyses using MSN and DAPC supported a geographical isolation of T. gondii populations of type II lineage, with limited migrations of strains between Tunisia and other countries of the Mediterranean basin.
However, the rare genetic proximities highlighted in our analyses (Fig. 3c) could correspond to geographical proximities (Algeria) or exchanges related to the colonial history of Tunisia from the Ottoman Empire (Turkey) to the French colonization.
Additional sampling efforts in countries neighboring Tunisia such as Morocco and Algeria will be needed to have robust estimates of the inter-country circulation of T. gondii strains.
In our study, the three detected type III isolates belong to different regions located in the center of Tunisia (Sidi Bouzid region) and on the coast (cities of Monastir and Sousse). This proves the spread of this strain in Tunisia. According to previous studies, Type III was the second most widespread genotype in the world, and was found in most sampled African countries 24.
The detection of an Africa 4 strain isolated from a sheep for the first time in Tunisia confirms its spread across the African continent. Strains belonging to this lineage (Africa 4) have been repeatedly isolated from immune-suppressed African patients in France 24 and more recently in poultry and rodents from Senegal, West Africa 4,38. Other studies using RFLP markers showed the circulation of this lineage (RFLP genotypes of this lineage are known under the designation of ToxoDB # 20 and ToxoDB#137) in the Emirates, China, Sri Lanka as well as in East Africa 23,24,39. Its geographic distribution follows an East-West axis connecting Asia and Africa.
In conclusion, this study revealed that the diversity of T. gondii strains in domestic animals from Tunisia is very similar to the diversity previously described in North Africa. Type II lineage was predominant, followed by type III and Africa 4. Population genetic analyses supported extensive circulation of strains within the country, but limited inter-country migrations of strains in the Mediterranean basin region.
However, further studies are needed on samples from domestic and wild animals collected in other unexplored regions of Tunisia and in neighbour countries for a more accurate description of the circulation of T. gondii strains in this region and the possible role of sheep herds in the spread of this parasite.
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.
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).
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.
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.
Acknowledgments
This study was funded by the Tunisian Ministry of Higher Education and Scientific Research.
The authors would like to thank Toxoplasma Biological Resource Center (BRC)/Centre National de Référence (CNR) Toxoplasmose and their team for their warm reception and the help and guidance they provided during genotyping the isolated strains in this study. We would also like to thank M. Mabrouk Saï and M. Younes Souki, the slaughterhouse veterinarians in Gafsa and Monastir for their collaboration in sampling and providing information. We are grateful to the poultry owners who participated in this study.
We are also very grateful to Habib Mezhoud, for his technical assistance.
Author contributions
H.B., A.L. and I.L. Conceived and designed the experiments, A.L., I.L., K.P., H.R. and N.P. Performed the experiments, A.L., L.G. and I.L. Wrote the main manuscript text, L.G. Performed genetic clustering. Drafted the manuscript, All authors reviewed the manuscript.
Competing interest’s statement
The author(s) declare no competing interests.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.