Genotyping of Toxoplasma gondii samples from Dakar

1 Department of Laboratories Abass NDAO Hospital, Dakar, Senegal. 2 Faculty of Medicine, Pharmacy and Dentistry (FMPOS), Dakar, Laboratory of Medical Biochemistry., Dakar, Senegal 3 Department of Animal Biology, Faculty of Science and Technology, Dakar – Senegal 4 Department of Parasitology and Mycology Pharmacy and Dentistry (FMPOS), Faculty of Medicine, Dakar, Senegal. 5 National Reference Center for Toxoplasmosis, Laboratory of Parasitology and Mycology, Centre Hospitalier Universitaire Dupuytren, 2 avenue Martin Luther King, 87042 Limoges, France.


INTRODUCTION
Toxoplasma gondii (T.gondii) is the agent of a cosmopolitan anthropozoonosis: toxoplasmosis.This intracellular parasite maintains an optional heteroxenous cycle between cats (definitive hosts) and other warm-blooded animals (intermediate hosts).Toxoplasmosis is almost always asymptomatic but can be severe in immunocompromised individual or after congenital transmission.The medical and veterinary importance of toxoplasmosis drives for 50 years numerous epidemiological studies to identify the reservoirs and modes of transmission of the parasite (Try et al., 2000).The consumption of raw or undercooked meat containing cysts of the parasite and the ingestion of oocysts with fruits and contaminated with faeces of cats vegetables are the two main modes of contamination.More recently, the consumption of water contaminated with oocysts was identified as a risk factor for toxoplasmosis in Brazil (Bahia -Oliveira et al., 2003).Waterborne outbreaks have been causing symptomatic toxoplasmosis sometimes fatal Panama (Benenson and et al., 1982), in Canada (Bowie et al., 1997) and Brazil (Tavern, 2002).The seroprevalence of human toxoplasmosis varies according to geographical areas.In Europe, it is 30 to 50% in the majority of countries in central and west and becomes less than 30% in the north.Low prevalences are recorded in North America, Southeast Asia and some African countries (Niger, South Africa) (Try et al., 2000).The highest prevalence (> 60 %) occurs primarily among the countries bordering the Gulf of Guinea and Latin America.These differences are mainly due to the larger survival of oocysts in humid climates.There are few infections in areas where cats are absent (Dubey, et al., 1997).
Oocysts have a central role in transmission of the parasite because they infect humans directly or indirectly through animals for slaughter.From the perspective of assessing the risk of toxoplasmosis associated with oocysts, it is necessary to determine the prevalence of oocysts in the environment.This is only possible with methods specific and sensitive to detection because the probability of isolating oocysts in naturally contaminated random sample is very low.The study of prevalence of toxoplasmosis in herbivores is also an interesting way to indirectly assess the prevalence of oocysts in the environment.
The work presented here is part of our research for our doctoral thesis and is in quite recent concern to identify strains of T. gondii circulating in Dakar, Senegal among others.It describes the nature of strains isolated at Dakar compared to the reference strains using a technique borrowed from Dr. Daniel Ajenberg namely multiplex PCR strains of Toxoplasma.The objective was to identify T. gondii samples isolated in Dakar (Senegal).

MATERIALS AND METHODS
All animals were tested in advance by ADHS.The blood was collected in a dry tube: The jugular vein in sheep, cattle and chickens (after decapitation); the heart chamber for wild animals killed or euthanized (if serology could be made from cardio -thoracic fluid ).
The parasite was then investigated in animals infected by pepsin digestion of the brain and/or heart according to the method of Dubey (1998c).
It consists of the extraction of DNA after experimental infection of strains from human toxoplasmosis by pepsin digestion of the brain and/or heart according to the method of Dubey (1998c).Finally, a multilocus typing was done after amplification of sequences by multiplex PCR (Ajzenberg et al., 2005) (Figures 1 and 2) Genetic typing of isolates of toxoplasma was based on the analysis of allelic polymorphism of five microsatellite markers: Tub2, W35, TgM -A, B18 and B17 (Ajzenberg et al., 2002a(Ajzenberg et al., , 2004(Ajzenberg et al., , 2005)).Their combination allows a very resolutive typing; can highlight allelic recombination, not detected by the conventional method of typing SAG2 single locus by PCR -RFLP.Typing is performed after amplification of sequences by multiplex PCR (Ajzenberg et al., 2005).DNA was extracted from the brain or ascites fluid of mice infected with the animal isolates with the kit QIAamp ® DNA Mini Kit (Qiagen, Courtaboeuf,France).
The primers were synthesized by Applied Biosystems (Courtaboeuf, France).For each primer pair, one primer was coupled to the 6 -carboxyfluorescein ( 6FAM ) or hexachloro -6carboxyfluorescein (HEX) to the 5 'end to determine the length of the PCR products of automated sequencer.PCR was performed   Gain realized by a thermocycler GeneAmp ® PCR System 2700 (Applied Biosystems, Courtaboeuf) was: initial denaturation at 95°C for 15 min; 35 cycles of amplification: 94°C (30 s), 63°C (3 min) and 72°C (60 s)/cycle; final extension at 60°C for 30 min.The PCR products were visualized with a molecular weight marker V (Roche Diagnostics, Meylan, France), after electrophoresis on an agarose gel (2% w/v) containing ethidium bromide (Figure 1).Depending on the intensity of the observed bands, the PCR products were diluted to 1/15 in the deionized formamide before electrophoresis by an automatic sequencer . 1 µl of each diluted product was mixed with 0.5 mu.l of size marker fluorescent ROX GeneScan ® 500 (75-500 bp, Applied Biosystems, Courtaboeuf) and 23 mu.l of deionized formamide.This mixture was denatured and analyzed by gel electrophoresis POP4 polyacrylamide held in a capillary (47cm/50 microns) (Applied Biosystems, Courtaboeuf).The fluorescence emission was recorded by an automated sequencer ( 310 Abiprism collection 1.0 , Applied Biosystems, Courtaboeuf ) and analyzed by the GeneScan ® Analysis Software version 2.1 (Applied Biosystems, Courtaboeuf ) .
Figure 2 shows the migration profile obtained for one isolate of type II after electrophoresis of PCR products on an automatic sequencer.Each significant peak fluorescence was an allele and its length was in bp.A genotype was characterized by a particular combination of alleles.An allele is often common to several types, which demonstrates the need for multilocus analysis to study the genetic diversity of Toxoplasma.Genotyping analysis Strain typing was performed by using the length polymorphism of 6 microsatellite markers in a modified multiplex assay.Elsewhere, we described a multiplex polymerase chain reaction (PCR) for typing strains of T. gondii by the use of five microsatellite markers (TUB2, W35, TgM-A, B18 and B17).For this study, we added a sixth microsatellite marker to the multiplex assay (M33) which was located on chromosome IV.

RESULTS AND DISCUSSION
The study population consisted of 10 isolates from Dakar, addressed to CRB Toxoplasma to establish the genetic profile.It shows a predominance of genotype I/III or Africa 1 (90%) and I / II / III or Africa 2 (25%) (Table 1) as the lengths of their alleles were characterized using 15 microsatellite markers characteristics (Table 2).In our study, 10 from 10 isolates were recombinant type I / III or Africa 1 and one type I / II / III genotype or Africa 2, while isolates belonging to three clonally lines are minority (type III: 0/10, type II: 0/10 and type I:0/10).The corresponding isolates were isolated in France.These recombinant genotypes have typically a mixture of type I alleles with the alleles of type III.So, these few isolates indicate flow in Dakar (Senegal) recombinant I/III genotypes and I/II/III  The figures for conventional typing were adjusted after the sequencing of the W35, B17 and B18 markers; ( ), Allelic polymorphism of these three markers are expressed as relative to conventional typing T. gondii: 1 alleles.2, 3 are reserved for clonally lines I, II and III; allele 1 or 3 is that the type I and III share allele, allele 2 or 3 means that the Types II and III from the allele.The higher numbers correspond to unconventional alleles (atypical) and consider adjusting sequencing.(Ajzenberg et al., 2004;Ajzenberg et al, 2009); designated respectively in terms of Africa 1 and Africa 2. It may be that the genotype Africa 1, which was mainly detected in immunocompromised patients in Africa West and Central is a clonally line (Ajzenberg et al., 2009).This genotype I / III have been isolated from chickens in Brazil or Portugal in cases of congenital toxoplasmosis.This said genotype "African" could represent a new clonally lineage, as we suggested in the first part.The fact of finding the same genotype I / III in several Brazilian isolates advocates this hypothesis (Pena et al., 2008;Ajzenberg, 2006).Similarly, Dubey and colleagues had described two recombinant strains Polish I / II / III (Dubey et al., 2008a (Ajzenberg et al., 2009;Lehmann et al., 2006).So, it is important to remember that Ajzenberg technique allows better resolution in a single PCR and remains the most appropriate.Standardization of the technique could be surprises in these authors.These preliminary results are the only currently available, not necessarily trying to say that these are the only type recombinants circulating in Dakar because of the small sample, given the relatively high prevalence of the disease in women pregnant (45.81 ± 7.30% in women with 44.24 ± 7.58% in the pregnant) (Ndiaye, 2012, personal communication).It would be interesting to study in this direction on a more consistent sample taking into account the different clinical forms to be affirmative.

Conclusion
Genotypes found in Dakar (Senegal) are recombinant genotypes mainly Africa 1 or Type I/III and Africa 2 or Type I/II/III.These two types are kept in CRB Toxoplasma at Limoges as reference strains.However, given the small size of the study population, it would be premature to say that those are the only strains circulating in the country.It would be interesting to make a more consistent sampling from all hosts involved to characterize the genetic polymorphism of T. gondii in Senegal.Also, making a correlation between genotypes and finding different clinical forms encountered would be of vital importance to our country
the QIAGEN ® Multiplex PCR kit (Qiagen, Courtaboeuf , France): 0.04 mM of each primer (Roche Diagnostics, Meylan, France), 4 and 6 mu.lDNA mu.l of water were added to the Multiplex Master Mix provided by QIAGEN ( 1x final concentration ) in a total volume of 25 mu.l.

Table 1 .
Result by genotype found.