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Seroprevalence of Toxoplasma gondii and direct genotyping using minisequencing in free-range pigs in Burkina Faso
International Journal of Food Microbiology 230 (2016) 10–15
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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Seroprevalence of Toxoplasma gondii and direct genotyping using minisequencing in free-range pigs in Burkina Faso Sanata Bamba a, Lénaïg Halos b,1, Zékiba Tarnagda a,c, Alexandre Alanio d,e, Pauline Macé b, Sandrine Moukoury f, Ibrahim Sangaré a, Robert Guiguemdé a, Jean-Marc Costa f, Stéphane Bretagne d,e,⁎ a
Institut Supérieur des Sciences de la Santé (INSSA), Université Polytechnique de Bobo-Dioulasso, Burkina Faso JRU BIPAR ENV, ANSES, UPVM, Animal Health Laboratory, Maisons-Alfort, France Institut de Recherche en Sciences de la Santé (IRSS), Bobo-Dioulasso, Burkina Faso d Laboratoire de Parasitologie-Mycologie, Groupe hospitalier Saint Louis Lariboisière, Assistance Publique-Hôpitaux de Paris (APHP), Paris, France e Université Paris Diderot, Sorbonne Paris Cité, Paris, France f Laboratoire Cerba, Cergy-Pontoise, France b c
a r t i c l e
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Article history: Received 5 December 2015 Received in revised form 5 April 2016 Accepted 12 April 2016 Available online 16 April 2016 Keywords: Toxoplasma gondii Pig Burkina Faso Seroprevalence Meat juice Quantitative PCR B1 gene AF487550 Repeated element Genotyping Minisequencing analysis
a b s t r a c t Background: Swine are a major source of meat for humans. As such, they can play an important role in the epidemiology of human toxoplasmosis. Therefore, we performed an epidemiological study to determine the prevalence and genotypes of Toxoplasma gondii in Burkina Fasan swine. Methods: The prevalence of T. gondii infection was evaluated in a 3-month prospective study at the slaughterhouse of Bobo-Dioulasso, Burkina Faso. Anti-Toxoplasma IgG titers were determined on meat juices from pig diaphragms using a commercially available ELISA assay. The DNA was extracted from 25 mg of heart biopsies of seropositive animals (IgG ≥50% of the control) and the presence of T. gondii DNA was detected using a quantitative PCR assay. Genotyping was performed directly on DNA from PCR-positive biopsies using high-resolution melting and minisequencing analyses of the repeated B1 gene. Results: The prevalence of carcasses positive for anti-Toxoplasma IgG was 29% (87/300) with no difference according to sex and age in contrast to the village of origin (p = 0.018). Of the 87 seropositive animals, two were PCR positive (parasitic load at 64 and 128 parasites/mg of heart biopsy). Two new genotypes belonging to Type II and Type III and different from the genotypes previously described using minisequencing were identified. Conclusion: Our study provides the first T. gondii seroprevalence data in Burkina Fasan swine. In addition, this direct typing method suggests diversity of the T. gondii genotypes circulating in domestic animals in Burkina Faso. This needs to be confirmed on a wider sampling of subjects. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Human toxoplasmosis is due to the coccidian parasite Toxoplasma gondii. It is a food-borne disease transmitted by the ingestion of cysts contained in meat, or of oocysts on contaminated fruits and vegetables, or present in water. Toxoplasmosis is often asymptomatic but can lead to devastating diseases in immunocompromised individuals such as HIV positive patients, and fetuses. Pork is often considered to be lower risk than mutton for toxoplasmosis because it is often cooked more thoroughly (Dubey, 2009). This limits the risk of human transmission. However, in low resource countries, pork is the most consumed meat (http://www.fao.org/ag/againfo/themes/ en/meat/backgr_sources.html), and hogs can participate in local ⁎ Corresponding author at: Laboratoire de Parasitologie-Mycologie, Hôpital St Louis, 1 avenue Claude Vellefaux, Paris, France. E-mail address: [email protected] (S. Bretagne). 1 Present address: Merial, Lyon, France.
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.04.016 0168-1605/© 2016 Elsevier B.V. All rights reserved.
cycles involving other animals such as cats, rodents and chicken. Systems for routine diagnosis, monitoring or reporting of toxoplasmosis in pigs are often undeveloped (Dubey, 2009; Timbilfou et al., 2012). In addition, there is little information available on the epidemiology of toxoplasmosis. Thus, there is an urgent need to consolidate knowledge on toxoplasmosis—especially as the food supply becomes globalized with changes in culinary habits and increased international travel. Genotyping of T. gondii has revealed genetic diversity and three main lineages (Types I, II, and III). These are now grouped into 15 haplogroups in six major clades (Su et al., 2012). Genotype determination is relevant for epidemiological reasons since genotypes circulating in Europe and North America are different from those identified in other part of the world, and also for clinical reasons (Xiao and Yolken, 2015). Indeed, pathogenicity in mice and in humans is different according to the genotype related to polymorphism in proteins such as dense granule and rhoptry (Melo et al., 2011). Severe inflammation in ocular toxoplasmosis was found associated with infection by strains harboring specific allele of the ROP18 gene (Sánchez et al., 2014).
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Several genotyping methods have been developed. The PCR-RFLP assay (for Restriction Fragment Length Polymorphism) is proposed as a global genotyping system but is limited by agar gel technologies with issues in reproducibility, lack of computerization for exchangeability (Su et al., 2006), and low sensitivity when dealing with low parasite DNA loads in tissue resulting in a variable proportion of genotyped samples (Bacci et al., 2015; Belfort-Neto et al., 2007; Wang et al., 2012). Microsatellite markers have emerged a very convenient means of genotyping since they were first reported (Costa et al., 1997). Nowadays, up to 13 markers in two multiplex assays are proposed for genotyping (Mercier et al., 2010). As with PCR-RFLP, the main technical limitation of the microsatellites markers is their low sensitivity. Because the targeted loci are only single copy, only high parasitic loads can be efficiently amplified and genotyped. To overcome this limitation, we developed a typing system based on the polymorphism of the repeated B1 gene of T. gondii using highresolution melting (HRM) analysis and mini-sequencing (Costa et al., 2013, 2011). Therefore, to determine the prevalence of T. gondii infection in pigs in Burkina Faso and to know the circulating strains, a prospective survey was performed at the Bobo-Dioulasso abattoir. Serology was performed on pork juices as previously reported (Hill et al., 2006). This was confirmed via real-time quantitative PCR (qPCR) for T. gondii DNA in heart biopsies of seropositive animals. The qPCR targeting the DNA repeated element of T. gondii has already been validated for human diagnosis (Reischl et al., 2003). Genotyping was performed on the qPCR-positive biopsies as previously reported (Costa et al., 2013, 2011). 2. Methods 2.1. Sampling protocol Meat samples were collected between September 18th and December 12th 2008 at the slaughterhouse of Bobo-Dioulasso, the second largest city of Burkina Faso (813,610 inhabitants in 2014). The “Abattoir Frigorifique de Bobo-Dioulasso” (AFB) is the only authorized slaughterhouse of the city. According to a recent report, the pork meat produced at the AFB has increased 30% between 2001 and 2006 and represents 12% of the produced meat (Timbilfou et al., 2012). Most of the pigs (85%) come from surrounding villages whereas 15% come from intensive breeding in the city or in the close vicinity of the city (Timbilfou et al., 2012). Most of the production (89% of 672 tons) is consumed locally, mainly as baked pork, and 11% is exported (Timbilfou et al., 2012). The number of slaughtered pigs increases in November and December, the months chosen for our study (Timbilfou et al., 2012). During the study period, the veterinary meat inspection condemned 13 carcasses suspected of cysticercosis, which were not included in the study. We therefore collected 300 pig samples. The age, gender and village of origin of each animal were recorded. For each pig, at least 100 g of the diaphragm as well as 50 g of the heart were collected in individual plastic bags, refrigerated at 4 °C and kept frozen at −20 °C after transportation to the laboratory. The meat juice was obtained from diaphragms cut into small pieces and frozen overnight at −20 °C in a plastic bag. After thawing at room temperature, the meat juice was centrifuged 15 min at 4000 rpm (1800g) and collected with a pipette into a microtube as previously described (Halos et al., 2010). 2.2. Detection of IgG The ELISA test (ELISA ID Screen® Toxoplasmosis Indirect; ID VET, Montpellier, France) was performed on diaphragm fluids according to the manufacturer's instructions except that the dilution factor was 1:2 and not 1:10 due to weaker concentrations in body fluids versus sera (Halos et al., 2010). The positive control was T. gondii-positive polyclonal ovine serum diluted in a stabilizing solution. The threshold of positivity was a titer of anti-Toxoplasma IgG ≥50% of the control.
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2.3. Detection of T. gondii DNA by real-time quantitative PCR (qPCR) DNA was extracted from 25 mg of heart biopsy of seropositive pigs using the QIAmp DNA Mini Kit for tissue protocol (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Extracted DNA was eluted in 200 μl, and the DNA concentration was determined with a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The 260 nm/280 nm and 260 nm/ 230 nm absorbance ratios were recorded with an expected value above 1.80 (range = 1.80–2.20). The qPCR targeting a repetitive DNA fragment (GenBank access number AF487550) in T. gondii was carried out in a StepOne® Instrument (Applied Biosystems). Some modifications of the primers and probe were brought to our previous publication reporting Light-Cycler qPCR assays (Reischl et al., 2003) to take into account the present change for the Taqman format. The two primers (TG-CG10: 5′-ATCAGGACTGTAGATGAAGG CG-3′, and TG-CG11: 5′-TAGATCGCATTCCGGTGTCT-3′) and a hydrolysis probe (TG-CG12: 5′FAM-AGAAGATGTTTCCGGCTTGGCTGCTTTAMRA3′) were designed to amplify and detect a conserved 140-bp region. In silico studies showed that the amplified region was specific for T. gondii when analyzed with the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/). No perfect alignment was observed with other organisms. One to five mismatches were present in primers and probes especially when compared to the closely related protozoan Hammondia hammondi (Genbank entries EU493285.1, EU493284.1, EU493282.1 EU493281.1, EU493283.1, EU493280.1 and EU493279.1). Such a repetitive element has not been described to date for Neospora caninum. Serial dilutions of two reference strains of Type I (RH) and Type II (B7) with a known concentration of T. gondii tachyzoites were used to validate the qPCR assay. Standard curves were obtained using serial 10-fold dilutions in water of reference strain DNA from 2.105 to 20 tachyzoites per reaction. By plotting the quantitative cycle (Cq) against the input target quantity, we obtained the following figures: slope: −3.292; Y-intercept: 19.745; R2: 0.98; and efficiency: 101.2%. Five microliters of extracted DNA from heart biopsies was used per reaction. PCR was set up in a final volume of 20 μl with the Taqman Universal PCR Master Mix (Applied Biosystems, Courtaboeuf, France) containing uracil-N-glycosylase. Each primer and probe (Sigma, Paris, France) was used at 0.5 μM and 0.25 μM, respectively. The reaction mixture was initially incubated for 2 min at 50 °C followed by a 10-min step at 95 °C. Amplification was performed for 50 cycles of denaturation (95 °C for 15 s) and annealing/extension (65 °C for 1 min). The results were expressed as the mean Cq of the duplicates tested for each specimen. Lower Cq values correspond to higher concentrations of the target DNA. During each run, a T. gondii DNA concentration corresponding to 100 T. gondii tachyzoites was used as a positive control (expected Cq = 33), and the elution buffer for DNA extraction was the negative control. The results were considered to be positive when a significant fluorescent signal above the baseline was detected. For 20 qPCR negative samples, the same T. gondii DNA concentration corresponding to 100 of T. gondii tachyzoites was used for detection of PCR inhibitors. After adding T. gondii DNA to the DNAs extracted from these 20 qPCR negative samples, the amplification was performed as above. 2.4. T. gondii genotyping Genotyping of T. gondii was based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene as described elsewhere (Costa et al., 2013, 2011). Briefly, 8 SNPs were studied with the HRM analysis of PCR products followed by purification of the PCR products and minisequencing analysis using the SNaPshot Multiplex kit (Applied Biosystems, Courtaboeuf, France). The reactions were run on an ABI3130XL genetic analyzer and analyzed using the Genescan software. DNA from reference strains for each of the three main lineages—Type I (RH strain), Type II (B7 strain), and Type III (C5 strain)—were kindly
p Value Unknown
56 (18.7) 3 (1–37) 0.81 (25/31) 26 (46) 29 (9.7) 3 (1–6) 2.22 (20/9) 10 (34)
Sindo Samoroga
55 (18.3) 3 (1–13) 1.04 (28/27) 13 (27) 13 (4.3) 4 (2–13) 5.5 (11/2) 5 (38)
Niangelogo Ndorola
19 (6.3) 2 (2–25) 1.38 (11/8) 1 (5) 5 (1.7) 8 (2–13) 0.67 (2/3) 4 (80)
Kouroma Faramanan
57 (19.0) 4 (2–12) 0.9 (27/30) 14 (25) 10 (3.3) 3 (2–5) 0.67 (4/6) 4 (40)
Douna Djiguera Dandé Bobo-Dioulasso Total
⁎ For the 244 animals for which the origin is known.
This is the first serological study in pigs from different villages slaughtered at Bobo Dioulasso, Burkina Faso. Our results show a 29% (87/300) seropositivity of IgG specific for Toxoplasma. We used an ELISA kit licensed by ID VET, France, to detect T. gondii antibodies in pigs. This ELISA kit is not specific for a given animal species; it has already been used for epidemiological studies of many species including pigs and wild boars (Arko-Mensah et al., 2000; Halos et al., 2010; Opsteegh et al., 2010b; Prangé et al., 2009; Richomme et al., 2010). However, interpretation and generalization of the results should be cautious. Indeed, there is no general agreement among the techniques and
Geographical origin (village or city)
4. Discussion
Table 1 Age, sex ratio, and anti-Toxoplasma IgG ≥50% according to the geographical origin of the 300 pig carcasses studied.
Among the 300 pig carcasses (mean age: 4 months; range 1–37) studied, anti-Toxoplasma seropositivity was 29% (87/300) (Table 1). There was no significant difference between the prevalence in boars and sows, 25.3% (39/154) and 32.9% (48/146), respectively, or between the age and titers of anti-T. gondii IgG (r2 = 0.002). Considering the 244 animals for which the village of origin was known, there was a significant difference (p = 0.018) according to the geographical distribution of the seropositive (anti-Toxoplasma IgG ≥50%) animals (Table 1). The heart biopsies of the 87 seropositive animals were tested for the presence of T. gondii DNA. The DNA concentrations of the pig heart biopsies were 201 ± 96 ng/μl with a 260/208 ratio of 1.9 ± 0.05. No amplification was seen in the negative controls. To test for PCR inhibitors, 20 samples were added to the same T. gondii DNA quantity as the positive control. The Cq values were 33.5 ± 0.25, which is similar to the ones obtained with the positive control. This demonstrates the absence of PCR inhibitors. In these conditions, two animals were positive via qPCR (Pig 1: a 37-month old male of unknown origin; and Pig 2: a 3-month old male from Dandé village). The Cqs were 33 and 32, which corresponded to 64 and 128 parasites/mg in the heart biopsy, respectively. To place the positive animals in a hierarchical clustering, the genotypes obtained for the two qPCR positive animals were analyzed with the genotypes of 7 reference strains and 23 congenital infection strains (Costa et al., 2013). Our clustering was already shown to fit with the three major lineages (Types I, II, and III). Using the arbitrary threshold of 1.25, two clades were observed in lineages II (IIa, and IIb). One qPCR-positive animal (Pig 1) clustered in Clade IIb, along with one from French Guiana and one from La Réunion Island amniotic fluids (Fig. 1). The other genotype (Pig 2) clustered in Clade III along with the genotype of the reference strains C5, NIA from Africa, and REN (Fig. 1)—these were already shown to cluster in Clade III along with French Guiana, La Réunion Island, La Guadeloupe, and New Caledonia amniotic fluids (Costa et al., 2013). None of the two pig genotypes clustered with the Africa 1 strain (DJO strain).
19 (6.3) 4 (3–25) 1.38 (11/8) 1 (5)
3. Results
5 (1.7) 4 (3–4) 0.25 (1/4) 1 (20)
Data are presented as the mean ± standard error or median and range. Statistical analyses were performed using Prism v4.0 (GraphPAD Software, San Diego, CA). A value of p b 0.05 was considered to be statistically significant.
32 (10.7) 12 (2–25) 0.26 (6/26) 8 (25)
2.5. Statistical analysis
300 4 (1–37) 0.95 (146/154) 87 (29)
provided by Asis Khan (David Sibley laboratory). Strains from Africa [Africa 1 (RMS-2003-DJO, thereafter DJO) and Africa 2 (CCH-2004NIA, thereafter NIA)], and the Caribbean (CCH-2005-REN, thereafter REN) were purchased from the French National Center of Reference for toxoplasmosis. The genotypes of the qPCR-positive animals were therefore determined together with six reference strains (RH, B7, C5, DJO, NIA, and REN). Twenty-three representative genotypes have already been obtained from amniotic fluids (Costa et al., 2013).
b0.0001 0.003 0.018⁎
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n (%) Median age mo (range) Sex ratio (M/F) Anti-Toxoplasma IgG ≥50% n (%)
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Fig. 1. Hierarchical clustering of the matrix generated for the eight SNPs using minisequencing analysis for 2 B1-PCR positive pig samples, 23 B1-PCR positive AFs and six reference strains. Clusters corresponding to the well-known lineages I, II and III were delineated. Using the arbitrary threshold of 1.25, two additional clades could be separated in lineages II (IIa and IIb). The names of the samples, reference strains and the names of the clades are indicated on the right. Clustering was calculated based on Euclidian distance metrics and complete linkage.
cut-off, no standardized reference materials, and no quality control program (Dubey, 2009). In addition, discrepancies can also rely on storage conditions, hemolysis, microbial contamination, or repeated freezingthawing cycles. Our second technical choice was to use muscle fluids instead of serum samples. Using the same T. gondii ELISA assay, tissue fluids were found to be considerably less efficient than serum samples (Gamble et al., 2005). Therefore, our estimate for Toxoplasma seroprevalence is likely to be lower than if serum were used. Another limitation is the non-validation of the test in developing countries where multiparasitism is common. Although not specifically studied here, we highlight the high number of cysticercosis cases (n = 13 at the slaughterhouse of Bobo-Dioulasso). This is another major parasitic disease transmitted via pork in Burkina Faso (Carabin et al., 2015). Similarly, Sarcocystis spp. infections could be responsible for cross reactivity with T. gondii antigens (Damriyasa et al., 2004). However, this is a pitfall of every study dealing with free-range pigs in tropical countries. Nevertheless, we used a commercially available assay to assume a certain reproducibility and availability. T. gondii is rarely detected in confinement raised swine from developed countries (Dubey, 2009) although the development of organic farms can modify this general statement (Bacci et al., 2015; Dubey et al., 2012). In sub-Saharan countries and more specifically in Burkina Faso, pigs are mainly raised in a traditional manner in small farming communities (Carabin et al., 2015; Timbilfou et al., 2012). Except for one study from Côte d'Ivoire (Prangé et al., 2009), that reported a seroprevalence in pork samples of 8.8% [range, 8.2– 9.37], the prevalence was usually high, including 20–36% in Zimbabwe
(Hove et al., 2005), 29% in Nigeria (Onyiche and Ademola, 2015), 32% in Ethiopia (Gebremedhin et al., 2015), and 39% in Ghana (ArkoMensah et al., 2000). The present seroprevalence (29%) is similar confirming the importance of pigs as intermediate hosts of T. gondii. The high positivity rate is explained by traditional breeding with occasional backyard scavenging pigs (Arko-Mensah et al., 2000; Hove et al., 2005; Prangé et al., 2009). Interestingly, differences in seroprevalence in the present study are more linked to a specific village than to gender or age as previously suggested for Toxoplasma in Ghana (ArkoMensah et al., 2000), suggesting very location-specific epidemiology. For the detection of T. gondii DNA, we studied heart tissue because this is one of the most commonly infected sites (Esteban-Redondo and Innes, 1998). Only two heart biopsies of the 87 were qPCR positive. The detection of T. gondii tissue cysts in organs of large animals is difficult due to their heterogeneous distribution. Thus, little is known about tissue cyst density. Indeed, the commercial DNA extraction kit is designed for 25 mg of tissue. This cannot assume that T. gondii cysts were present in the processed biopsies. As a consequence, the most sensitive method for T. gondii detection in meat products is still the demonstration of the presence of the parasite by mouse bioassays because 50 g of meat can be processed (Dubey et al., 2012), whereas the quantity of meat amenable to DNA extraction is much lower (25 mg in the present study using a commercial kit). However, the higher sensitivity of bioassays could be challenged when using newer molecular tools with an initial DNA enrichment (Gomez-Samblas et al., 2015; Opsteegh et al., 2010a). The present minisequencing method was developed to genotype even in the presence of low amounts of parasite DNA (Costa et al.,
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2013, 2011). Indeed, the amount of T. gondii DNA obtained from the biopsies prevented any direct microsatellite typing although minisequencing allowed the genotyping of T. gondii from the two positive samples. Thus, one identified genotype was placed into Clade III along with other reference strains and amniotic fluids from very different countries such as French Guiana, La Réunion, Guadeloupe, New Caledonia islands, and France (Costa et al., 2013), corresponding to the worldwide Type III genotype (Mercier et al., 2010; Su et al., 2012). The second pig had a different genotype and was placed in Clade IIb with amniotic fluids from French Guiana and La Réunion Island (Costa et al., 2013), corresponding to the worldwide Type II genotype (Mercier et al., 2010; Su et al., 2012). The detection of two different genotypes not strictly identical to already reported genotypes using our minisequencing method from such a limited geographical area suggests an unusual diversity in the circulating genotypes in Africa compared to the situation in Europe or USA as already reported (Xiao and Yolken, 2015). At the village level, the number of PCR-positive animals precludes any meaningful deciphering between a local genotype and the circulation of several genotypes. The presence of more than one clonal genotype in circulation in ranging pigs from three organically managed farms in northern Italy has already been reported using nested PCRRFLP typing (Bacci et al., 2015). In contrast, two different clones after bioassays were identified in two organic farms in Michigan (Dubey et al., 2012). The hypothesis of local epidemiology, whatever the circulating genotype, is also supported in our study by the heterogeneous distribution of seropositive animals according to the village. Of note, no swine clustered with the Africa 1 (DJO) strain, which has been reported to be more virulent in mice (Mercier et al., 2010). The minisequencing method, nevertheless, presents some limitations. The number of genetic markers (n = 8) is lower than the system based on 13 microsatellite markers (Mercier et al., 2010). The SNPs are also all in the same locus, the B1 gene—in contrast to the microsatellites markers which are more evenly spread across the genome. Studying one locus can introduce bias usually resulting in an underestimation of genetic differences. Therefore, a strict comparison with the genotypes reported in Africa using microsatellite markers (Mercier et al., 2010) was not possible. The main advantage of our system over microsatellite markers is the avoidance of a bioassay designed to amplify live parasites by inoculation in mice. This bioassay introduces bias when all animals from a given species are not all positive (Dubey et al., 2012; Mercier et al., 2010) or when T. gondii from a given intermediate host are not viable in mice (Aroussi et al., 2015). To understand the role of free-range pigs in the epidemiology of local toxoplasmosis and more specifically for human infection, more information is needed about the genotypes infecting the local populations. The seropositivity of animals strongly correlates with live, infective tissue cysts in meat (Dubey, 2009). Therefore, seropositive pork could be the source of human infection either directly, although this meat is either not consumed for religious beliefs or always consumed very well cooked in Burkina Faso, or indirectly if raw meat is consumed by feral cats in the vicinity of the slaughterhouse. The other method of human infection is indeed the ingestion of oocysts excreted by cats and the role of other intermediate hosts cannot be excluded. Poor sanitation could be a cause of contamination of the food by oocysts although oocysts are known to be rapidly killed at temperatures N 45 °C (Dubey, 1998)—this is often reached in summer months in Burkina Faso. Incidentally, if ingestion of environmental oocysts does not occur during the summer, the pigs are not progressively infected with increasing age and this can explain, at least in part, the lack of correlation between Toxoplasma seroprevalence in pigs and age. In addition, freeranging pigs live adjacent with human beings and are a sentinel of infection for the human population. The high prevalence of infection in swine reported here echoes what is seen in humans. Indeed, whatever the source of human infection, the Toxoplasma-seroprevalence in pregnant women is 33.1% at Bobo-Dioulasso. This suggests that maternal transmission to the fetus is possible (Bamba et al., 2014).
5. Conclusion The high rate of Toxoplasma seropositivity in Burkina Fasan freerange pigs highlights the need to monitor the spread of the parasite and the need to reduce its transmission. In addition, our study revealed variability of the genotypes circulating in small geographic areas, underlining the need for further genotype studies for epidemiological purposes. Genotyping of human cases in Burkina should help elucidate the source of infection.
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Halos, L., Thébault, A., Aubert, D., Thomas, M., Perret, C., Geers, R., Alliot, A., Escotte-Binet, S., Ajzenberg, D., Dardé, M.-L., Durand, B., Boireau, P., Villena, I., 2010. An innovative survey underlining the significant level of contamination by Toxoplasma gondii of ovine meat consumed in France. Int. J. Parasitol. 40, 193–200. Hill, D.E., Chirukandoth, S., Dubey, J.P., Lunney, J.K., Gamble, H.R., 2006. Comparison of detection methods for Toxoplasma gondii in naturally and experimentally infected swine. Vet. Parasitol. 141, 9–17. Hove, T., Lind, P., Mukaratirwa, S., 2005. Seroprevalence of Toxoplasma gondii infection in domestic pigs reared under different management systems in Zimbabwe. Onderstepoort J. Vet. Res. 72, 231–237. Melo, M.B., Jensen, K.D.C., Saeij, J.P.J., 2011. Toxoplasma gondii effectors are master regulators of the inflammatory response. Trends Parasitol. 27, 487–495. Mercier, A., Devillard, S., Ngoubangoye, B., Bonnabau, H., Bañuls, A.-L., Durand, P., Salle, B., Ajzenberg, D., Dardé, M.-L., 2010. Additional haplogroups of Toxoplasma gondii out of Africa: population structure and mouse-virulence of strains from Gabon. PLoS Negl. Trop. Dis. 4, e876. Onyiche, T.E., Ademola, I.O., 2015. Seroprevalence of anti-Toxoplasma gondii antibodies in cattle and pigs in Ibadan, Nigeria. J. Parasit. Dis. 39, 309–314.
S. Bamba et al. / International Journal of Food Microbiology 230 (2016) 10–15 Opsteegh, M., Langelaar, M., Sprong, H., Hartog den, L., De Craeye, S., Bokken, G., Ajzenberg, D., Kijlstra, A., van der Giessen, J., 2010a. Direct detection and genotyping of Toxoplasma gondii in meat samples using magnetic capture and PCR. Int. J. Food Microbiol. 139, 193–201. Opsteegh, M., Teunis, P., Mensink, M., Züchner, L., Titilincu, A., Langelaar, M., van der Giessen, J., 2010b. Evaluation of ELISA test characteristics and estimation of Toxoplasma gondii seroprevalence in Dutch sheep using mixture models. Prev. Vet. Med. 96, 232–240. Prangé, A., Perret, C., Marié, J.L., Calvet, F., Halos, L., Boireau, P., Davoust, B., 2009. [Toxoplasmosis: a survey on meat products in Côte d'Ivoire]. Med. Trop. (Mars) 69, 629–630. Reischl, U., Bretagne, S., Krüger, D., Ernault, P., Costa, J.-M., 2003. Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect. Dis. 3, 7. Richomme, C., Afonso, E., Tolon, V., Ducrot, C., Halos, L., Alliot, A., Perret, C., Thomas, M., Boireau, P., Gilot-Fromont, E., 2010. Seroprevalence and factors associated with Toxoplasma gondii infection in wild boar (Sus scrofa) in a Mediterranean island. Epidemiol. Infect. 138, 1257–1266.
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Sánchez, V., de-la-Torre, A., Gómez-Marín, J.E., 2014. Characterization of ROP18 alleles in human toxoplasmosis. Parasitol. Int. 63, 463–469. Su, C., Zhang, X., Dubey, J.P., 2006. Genotyping of Toxoplasma gondii by multilocus PCRRFLP markers: a high resolution and simple method for identification of parasites. Int. J. Parasitol. 36, 841–848. Su, C., Khan, A., Zhou, P., Majumdar, D., Ajzenberg, D., Dardé, M.-L., Zhu, X.-Q., Ajioka, J.W., Rosenthal, B.M., Dubey, J.P., Sibley, L.D., 2012. Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proc. Natl. Acad. Sci. U. S. A. 109, 5844–5849. Timbilfou, K., Youssouf, M.L., Richard, K.S., Chantal-Yvette, K.-Z., 2012. Approvisionnement en porcs vifs et viande porcine de la ville de Bobo-Dioulasso (Burkina Faso). J. Agric. Environ. Int. Dev. (JAEID) 106, 105–122. Wang, H., Wang, T., Luo, Q., Huo, X., Wang, L., Liu, T., Xu, X., Wang, Y., Lu, F., Lun, Z., Yu, L., Shen, J., 2012. Prevalence and genotypes of Toxoplasma gondii in pork from retail meat stores in Eastern China. Int. J. Food Microbiol. 157, 393–397. Xiao, J., Yolken, R.H., 2015. Strain hypothesis of Toxoplasma gondii infection on the outcome of human diseases. Acta Physiol (Oxford) 213, 828–845.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Seroprevalence of Toxoplasma gondii and direct genotyping using minisequencing in free-range pigs in Burkina Faso Sanata Bamba a, Lénaïg Halos b,1, Zékiba Tarnagda a,c, Alexandre Alanio d,e, Pauline Macé b, Sandrine Moukoury f, Ibrahim Sangaré a, Robert Guiguemdé a, Jean-Marc Costa f, Stéphane Bretagne d,e,⁎ a
Institut Supérieur des Sciences de la Santé (INSSA), Université Polytechnique de Bobo-Dioulasso, Burkina Faso JRU BIPAR ENV, ANSES, UPVM, Animal Health Laboratory, Maisons-Alfort, France Institut de Recherche en Sciences de la Santé (IRSS), Bobo-Dioulasso, Burkina Faso d Laboratoire de Parasitologie-Mycologie, Groupe hospitalier Saint Louis Lariboisière, Assistance Publique-Hôpitaux de Paris (APHP), Paris, France e Université Paris Diderot, Sorbonne Paris Cité, Paris, France f Laboratoire Cerba, Cergy-Pontoise, France b c
a r t i c l e
i n f o
Article history: Received 5 December 2015 Received in revised form 5 April 2016 Accepted 12 April 2016 Available online 16 April 2016 Keywords: Toxoplasma gondii Pig Burkina Faso Seroprevalence Meat juice Quantitative PCR B1 gene AF487550 Repeated element Genotyping Minisequencing analysis
a b s t r a c t Background: Swine are a major source of meat for humans. As such, they can play an important role in the epidemiology of human toxoplasmosis. Therefore, we performed an epidemiological study to determine the prevalence and genotypes of Toxoplasma gondii in Burkina Fasan swine. Methods: The prevalence of T. gondii infection was evaluated in a 3-month prospective study at the slaughterhouse of Bobo-Dioulasso, Burkina Faso. Anti-Toxoplasma IgG titers were determined on meat juices from pig diaphragms using a commercially available ELISA assay. The DNA was extracted from 25 mg of heart biopsies of seropositive animals (IgG ≥50% of the control) and the presence of T. gondii DNA was detected using a quantitative PCR assay. Genotyping was performed directly on DNA from PCR-positive biopsies using high-resolution melting and minisequencing analyses of the repeated B1 gene. Results: The prevalence of carcasses positive for anti-Toxoplasma IgG was 29% (87/300) with no difference according to sex and age in contrast to the village of origin (p = 0.018). Of the 87 seropositive animals, two were PCR positive (parasitic load at 64 and 128 parasites/mg of heart biopsy). Two new genotypes belonging to Type II and Type III and different from the genotypes previously described using minisequencing were identified. Conclusion: Our study provides the first T. gondii seroprevalence data in Burkina Fasan swine. In addition, this direct typing method suggests diversity of the T. gondii genotypes circulating in domestic animals in Burkina Faso. This needs to be confirmed on a wider sampling of subjects. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Human toxoplasmosis is due to the coccidian parasite Toxoplasma gondii. It is a food-borne disease transmitted by the ingestion of cysts contained in meat, or of oocysts on contaminated fruits and vegetables, or present in water. Toxoplasmosis is often asymptomatic but can lead to devastating diseases in immunocompromised individuals such as HIV positive patients, and fetuses. Pork is often considered to be lower risk than mutton for toxoplasmosis because it is often cooked more thoroughly (Dubey, 2009). This limits the risk of human transmission. However, in low resource countries, pork is the most consumed meat (http://www.fao.org/ag/againfo/themes/ en/meat/backgr_sources.html), and hogs can participate in local ⁎ Corresponding author at: Laboratoire de Parasitologie-Mycologie, Hôpital St Louis, 1 avenue Claude Vellefaux, Paris, France. E-mail address: [email protected] (S. Bretagne). 1 Present address: Merial, Lyon, France.
http://dx.doi.org/10.1016/j.ijfoodmicro.2016.04.016 0168-1605/© 2016 Elsevier B.V. All rights reserved.
cycles involving other animals such as cats, rodents and chicken. Systems for routine diagnosis, monitoring or reporting of toxoplasmosis in pigs are often undeveloped (Dubey, 2009; Timbilfou et al., 2012). In addition, there is little information available on the epidemiology of toxoplasmosis. Thus, there is an urgent need to consolidate knowledge on toxoplasmosis—especially as the food supply becomes globalized with changes in culinary habits and increased international travel. Genotyping of T. gondii has revealed genetic diversity and three main lineages (Types I, II, and III). These are now grouped into 15 haplogroups in six major clades (Su et al., 2012). Genotype determination is relevant for epidemiological reasons since genotypes circulating in Europe and North America are different from those identified in other part of the world, and also for clinical reasons (Xiao and Yolken, 2015). Indeed, pathogenicity in mice and in humans is different according to the genotype related to polymorphism in proteins such as dense granule and rhoptry (Melo et al., 2011). Severe inflammation in ocular toxoplasmosis was found associated with infection by strains harboring specific allele of the ROP18 gene (Sánchez et al., 2014).
S. Bamba et al. / International Journal of Food Microbiology 230 (2016) 10–15
Several genotyping methods have been developed. The PCR-RFLP assay (for Restriction Fragment Length Polymorphism) is proposed as a global genotyping system but is limited by agar gel technologies with issues in reproducibility, lack of computerization for exchangeability (Su et al., 2006), and low sensitivity when dealing with low parasite DNA loads in tissue resulting in a variable proportion of genotyped samples (Bacci et al., 2015; Belfort-Neto et al., 2007; Wang et al., 2012). Microsatellite markers have emerged a very convenient means of genotyping since they were first reported (Costa et al., 1997). Nowadays, up to 13 markers in two multiplex assays are proposed for genotyping (Mercier et al., 2010). As with PCR-RFLP, the main technical limitation of the microsatellites markers is their low sensitivity. Because the targeted loci are only single copy, only high parasitic loads can be efficiently amplified and genotyped. To overcome this limitation, we developed a typing system based on the polymorphism of the repeated B1 gene of T. gondii using highresolution melting (HRM) analysis and mini-sequencing (Costa et al., 2013, 2011). Therefore, to determine the prevalence of T. gondii infection in pigs in Burkina Faso and to know the circulating strains, a prospective survey was performed at the Bobo-Dioulasso abattoir. Serology was performed on pork juices as previously reported (Hill et al., 2006). This was confirmed via real-time quantitative PCR (qPCR) for T. gondii DNA in heart biopsies of seropositive animals. The qPCR targeting the DNA repeated element of T. gondii has already been validated for human diagnosis (Reischl et al., 2003). Genotyping was performed on the qPCR-positive biopsies as previously reported (Costa et al., 2013, 2011). 2. Methods 2.1. Sampling protocol Meat samples were collected between September 18th and December 12th 2008 at the slaughterhouse of Bobo-Dioulasso, the second largest city of Burkina Faso (813,610 inhabitants in 2014). The “Abattoir Frigorifique de Bobo-Dioulasso” (AFB) is the only authorized slaughterhouse of the city. According to a recent report, the pork meat produced at the AFB has increased 30% between 2001 and 2006 and represents 12% of the produced meat (Timbilfou et al., 2012). Most of the pigs (85%) come from surrounding villages whereas 15% come from intensive breeding in the city or in the close vicinity of the city (Timbilfou et al., 2012). Most of the production (89% of 672 tons) is consumed locally, mainly as baked pork, and 11% is exported (Timbilfou et al., 2012). The number of slaughtered pigs increases in November and December, the months chosen for our study (Timbilfou et al., 2012). During the study period, the veterinary meat inspection condemned 13 carcasses suspected of cysticercosis, which were not included in the study. We therefore collected 300 pig samples. The age, gender and village of origin of each animal were recorded. For each pig, at least 100 g of the diaphragm as well as 50 g of the heart were collected in individual plastic bags, refrigerated at 4 °C and kept frozen at −20 °C after transportation to the laboratory. The meat juice was obtained from diaphragms cut into small pieces and frozen overnight at −20 °C in a plastic bag. After thawing at room temperature, the meat juice was centrifuged 15 min at 4000 rpm (1800g) and collected with a pipette into a microtube as previously described (Halos et al., 2010). 2.2. Detection of IgG The ELISA test (ELISA ID Screen® Toxoplasmosis Indirect; ID VET, Montpellier, France) was performed on diaphragm fluids according to the manufacturer's instructions except that the dilution factor was 1:2 and not 1:10 due to weaker concentrations in body fluids versus sera (Halos et al., 2010). The positive control was T. gondii-positive polyclonal ovine serum diluted in a stabilizing solution. The threshold of positivity was a titer of anti-Toxoplasma IgG ≥50% of the control.
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2.3. Detection of T. gondii DNA by real-time quantitative PCR (qPCR) DNA was extracted from 25 mg of heart biopsy of seropositive pigs using the QIAmp DNA Mini Kit for tissue protocol (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. Extracted DNA was eluted in 200 μl, and the DNA concentration was determined with a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The 260 nm/280 nm and 260 nm/ 230 nm absorbance ratios were recorded with an expected value above 1.80 (range = 1.80–2.20). The qPCR targeting a repetitive DNA fragment (GenBank access number AF487550) in T. gondii was carried out in a StepOne® Instrument (Applied Biosystems). Some modifications of the primers and probe were brought to our previous publication reporting Light-Cycler qPCR assays (Reischl et al., 2003) to take into account the present change for the Taqman format. The two primers (TG-CG10: 5′-ATCAGGACTGTAGATGAAGG CG-3′, and TG-CG11: 5′-TAGATCGCATTCCGGTGTCT-3′) and a hydrolysis probe (TG-CG12: 5′FAM-AGAAGATGTTTCCGGCTTGGCTGCTTTAMRA3′) were designed to amplify and detect a conserved 140-bp region. In silico studies showed that the amplified region was specific for T. gondii when analyzed with the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/). No perfect alignment was observed with other organisms. One to five mismatches were present in primers and probes especially when compared to the closely related protozoan Hammondia hammondi (Genbank entries EU493285.1, EU493284.1, EU493282.1 EU493281.1, EU493283.1, EU493280.1 and EU493279.1). Such a repetitive element has not been described to date for Neospora caninum. Serial dilutions of two reference strains of Type I (RH) and Type II (B7) with a known concentration of T. gondii tachyzoites were used to validate the qPCR assay. Standard curves were obtained using serial 10-fold dilutions in water of reference strain DNA from 2.105 to 20 tachyzoites per reaction. By plotting the quantitative cycle (Cq) against the input target quantity, we obtained the following figures: slope: −3.292; Y-intercept: 19.745; R2: 0.98; and efficiency: 101.2%. Five microliters of extracted DNA from heart biopsies was used per reaction. PCR was set up in a final volume of 20 μl with the Taqman Universal PCR Master Mix (Applied Biosystems, Courtaboeuf, France) containing uracil-N-glycosylase. Each primer and probe (Sigma, Paris, France) was used at 0.5 μM and 0.25 μM, respectively. The reaction mixture was initially incubated for 2 min at 50 °C followed by a 10-min step at 95 °C. Amplification was performed for 50 cycles of denaturation (95 °C for 15 s) and annealing/extension (65 °C for 1 min). The results were expressed as the mean Cq of the duplicates tested for each specimen. Lower Cq values correspond to higher concentrations of the target DNA. During each run, a T. gondii DNA concentration corresponding to 100 T. gondii tachyzoites was used as a positive control (expected Cq = 33), and the elution buffer for DNA extraction was the negative control. The results were considered to be positive when a significant fluorescent signal above the baseline was detected. For 20 qPCR negative samples, the same T. gondii DNA concentration corresponding to 100 of T. gondii tachyzoites was used for detection of PCR inhibitors. After adding T. gondii DNA to the DNAs extracted from these 20 qPCR negative samples, the amplification was performed as above. 2.4. T. gondii genotyping Genotyping of T. gondii was based on an analysis of eight nucleotide polymorphisms (SNPs) located within the B1 gene as described elsewhere (Costa et al., 2013, 2011). Briefly, 8 SNPs were studied with the HRM analysis of PCR products followed by purification of the PCR products and minisequencing analysis using the SNaPshot Multiplex kit (Applied Biosystems, Courtaboeuf, France). The reactions were run on an ABI3130XL genetic analyzer and analyzed using the Genescan software. DNA from reference strains for each of the three main lineages—Type I (RH strain), Type II (B7 strain), and Type III (C5 strain)—were kindly
p Value Unknown
56 (18.7) 3 (1–37) 0.81 (25/31) 26 (46) 29 (9.7) 3 (1–6) 2.22 (20/9) 10 (34)
Sindo Samoroga
55 (18.3) 3 (1–13) 1.04 (28/27) 13 (27) 13 (4.3) 4 (2–13) 5.5 (11/2) 5 (38)
Niangelogo Ndorola
19 (6.3) 2 (2–25) 1.38 (11/8) 1 (5) 5 (1.7) 8 (2–13) 0.67 (2/3) 4 (80)
Kouroma Faramanan
57 (19.0) 4 (2–12) 0.9 (27/30) 14 (25) 10 (3.3) 3 (2–5) 0.67 (4/6) 4 (40)
Douna Djiguera Dandé Bobo-Dioulasso Total
⁎ For the 244 animals for which the origin is known.
This is the first serological study in pigs from different villages slaughtered at Bobo Dioulasso, Burkina Faso. Our results show a 29% (87/300) seropositivity of IgG specific for Toxoplasma. We used an ELISA kit licensed by ID VET, France, to detect T. gondii antibodies in pigs. This ELISA kit is not specific for a given animal species; it has already been used for epidemiological studies of many species including pigs and wild boars (Arko-Mensah et al., 2000; Halos et al., 2010; Opsteegh et al., 2010b; Prangé et al., 2009; Richomme et al., 2010). However, interpretation and generalization of the results should be cautious. Indeed, there is no general agreement among the techniques and
Geographical origin (village or city)
4. Discussion
Table 1 Age, sex ratio, and anti-Toxoplasma IgG ≥50% according to the geographical origin of the 300 pig carcasses studied.
Among the 300 pig carcasses (mean age: 4 months; range 1–37) studied, anti-Toxoplasma seropositivity was 29% (87/300) (Table 1). There was no significant difference between the prevalence in boars and sows, 25.3% (39/154) and 32.9% (48/146), respectively, or between the age and titers of anti-T. gondii IgG (r2 = 0.002). Considering the 244 animals for which the village of origin was known, there was a significant difference (p = 0.018) according to the geographical distribution of the seropositive (anti-Toxoplasma IgG ≥50%) animals (Table 1). The heart biopsies of the 87 seropositive animals were tested for the presence of T. gondii DNA. The DNA concentrations of the pig heart biopsies were 201 ± 96 ng/μl with a 260/208 ratio of 1.9 ± 0.05. No amplification was seen in the negative controls. To test for PCR inhibitors, 20 samples were added to the same T. gondii DNA quantity as the positive control. The Cq values were 33.5 ± 0.25, which is similar to the ones obtained with the positive control. This demonstrates the absence of PCR inhibitors. In these conditions, two animals were positive via qPCR (Pig 1: a 37-month old male of unknown origin; and Pig 2: a 3-month old male from Dandé village). The Cqs were 33 and 32, which corresponded to 64 and 128 parasites/mg in the heart biopsy, respectively. To place the positive animals in a hierarchical clustering, the genotypes obtained for the two qPCR positive animals were analyzed with the genotypes of 7 reference strains and 23 congenital infection strains (Costa et al., 2013). Our clustering was already shown to fit with the three major lineages (Types I, II, and III). Using the arbitrary threshold of 1.25, two clades were observed in lineages II (IIa, and IIb). One qPCR-positive animal (Pig 1) clustered in Clade IIb, along with one from French Guiana and one from La Réunion Island amniotic fluids (Fig. 1). The other genotype (Pig 2) clustered in Clade III along with the genotype of the reference strains C5, NIA from Africa, and REN (Fig. 1)—these were already shown to cluster in Clade III along with French Guiana, La Réunion Island, La Guadeloupe, and New Caledonia amniotic fluids (Costa et al., 2013). None of the two pig genotypes clustered with the Africa 1 strain (DJO strain).
19 (6.3) 4 (3–25) 1.38 (11/8) 1 (5)
3. Results
5 (1.7) 4 (3–4) 0.25 (1/4) 1 (20)
Data are presented as the mean ± standard error or median and range. Statistical analyses were performed using Prism v4.0 (GraphPAD Software, San Diego, CA). A value of p b 0.05 was considered to be statistically significant.
32 (10.7) 12 (2–25) 0.26 (6/26) 8 (25)
2.5. Statistical analysis
300 4 (1–37) 0.95 (146/154) 87 (29)
provided by Asis Khan (David Sibley laboratory). Strains from Africa [Africa 1 (RMS-2003-DJO, thereafter DJO) and Africa 2 (CCH-2004NIA, thereafter NIA)], and the Caribbean (CCH-2005-REN, thereafter REN) were purchased from the French National Center of Reference for toxoplasmosis. The genotypes of the qPCR-positive animals were therefore determined together with six reference strains (RH, B7, C5, DJO, NIA, and REN). Twenty-three representative genotypes have already been obtained from amniotic fluids (Costa et al., 2013).
b0.0001 0.003 0.018⁎
S. Bamba et al. / International Journal of Food Microbiology 230 (2016) 10–15
n (%) Median age mo (range) Sex ratio (M/F) Anti-Toxoplasma IgG ≥50% n (%)
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Fig. 1. Hierarchical clustering of the matrix generated for the eight SNPs using minisequencing analysis for 2 B1-PCR positive pig samples, 23 B1-PCR positive AFs and six reference strains. Clusters corresponding to the well-known lineages I, II and III were delineated. Using the arbitrary threshold of 1.25, two additional clades could be separated in lineages II (IIa and IIb). The names of the samples, reference strains and the names of the clades are indicated on the right. Clustering was calculated based on Euclidian distance metrics and complete linkage.
cut-off, no standardized reference materials, and no quality control program (Dubey, 2009). In addition, discrepancies can also rely on storage conditions, hemolysis, microbial contamination, or repeated freezingthawing cycles. Our second technical choice was to use muscle fluids instead of serum samples. Using the same T. gondii ELISA assay, tissue fluids were found to be considerably less efficient than serum samples (Gamble et al., 2005). Therefore, our estimate for Toxoplasma seroprevalence is likely to be lower than if serum were used. Another limitation is the non-validation of the test in developing countries where multiparasitism is common. Although not specifically studied here, we highlight the high number of cysticercosis cases (n = 13 at the slaughterhouse of Bobo-Dioulasso). This is another major parasitic disease transmitted via pork in Burkina Faso (Carabin et al., 2015). Similarly, Sarcocystis spp. infections could be responsible for cross reactivity with T. gondii antigens (Damriyasa et al., 2004). However, this is a pitfall of every study dealing with free-range pigs in tropical countries. Nevertheless, we used a commercially available assay to assume a certain reproducibility and availability. T. gondii is rarely detected in confinement raised swine from developed countries (Dubey, 2009) although the development of organic farms can modify this general statement (Bacci et al., 2015; Dubey et al., 2012). In sub-Saharan countries and more specifically in Burkina Faso, pigs are mainly raised in a traditional manner in small farming communities (Carabin et al., 2015; Timbilfou et al., 2012). Except for one study from Côte d'Ivoire (Prangé et al., 2009), that reported a seroprevalence in pork samples of 8.8% [range, 8.2– 9.37], the prevalence was usually high, including 20–36% in Zimbabwe
(Hove et al., 2005), 29% in Nigeria (Onyiche and Ademola, 2015), 32% in Ethiopia (Gebremedhin et al., 2015), and 39% in Ghana (ArkoMensah et al., 2000). The present seroprevalence (29%) is similar confirming the importance of pigs as intermediate hosts of T. gondii. The high positivity rate is explained by traditional breeding with occasional backyard scavenging pigs (Arko-Mensah et al., 2000; Hove et al., 2005; Prangé et al., 2009). Interestingly, differences in seroprevalence in the present study are more linked to a specific village than to gender or age as previously suggested for Toxoplasma in Ghana (ArkoMensah et al., 2000), suggesting very location-specific epidemiology. For the detection of T. gondii DNA, we studied heart tissue because this is one of the most commonly infected sites (Esteban-Redondo and Innes, 1998). Only two heart biopsies of the 87 were qPCR positive. The detection of T. gondii tissue cysts in organs of large animals is difficult due to their heterogeneous distribution. Thus, little is known about tissue cyst density. Indeed, the commercial DNA extraction kit is designed for 25 mg of tissue. This cannot assume that T. gondii cysts were present in the processed biopsies. As a consequence, the most sensitive method for T. gondii detection in meat products is still the demonstration of the presence of the parasite by mouse bioassays because 50 g of meat can be processed (Dubey et al., 2012), whereas the quantity of meat amenable to DNA extraction is much lower (25 mg in the present study using a commercial kit). However, the higher sensitivity of bioassays could be challenged when using newer molecular tools with an initial DNA enrichment (Gomez-Samblas et al., 2015; Opsteegh et al., 2010a). The present minisequencing method was developed to genotype even in the presence of low amounts of parasite DNA (Costa et al.,
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2013, 2011). Indeed, the amount of T. gondii DNA obtained from the biopsies prevented any direct microsatellite typing although minisequencing allowed the genotyping of T. gondii from the two positive samples. Thus, one identified genotype was placed into Clade III along with other reference strains and amniotic fluids from very different countries such as French Guiana, La Réunion, Guadeloupe, New Caledonia islands, and France (Costa et al., 2013), corresponding to the worldwide Type III genotype (Mercier et al., 2010; Su et al., 2012). The second pig had a different genotype and was placed in Clade IIb with amniotic fluids from French Guiana and La Réunion Island (Costa et al., 2013), corresponding to the worldwide Type II genotype (Mercier et al., 2010; Su et al., 2012). The detection of two different genotypes not strictly identical to already reported genotypes using our minisequencing method from such a limited geographical area suggests an unusual diversity in the circulating genotypes in Africa compared to the situation in Europe or USA as already reported (Xiao and Yolken, 2015). At the village level, the number of PCR-positive animals precludes any meaningful deciphering between a local genotype and the circulation of several genotypes. The presence of more than one clonal genotype in circulation in ranging pigs from three organically managed farms in northern Italy has already been reported using nested PCRRFLP typing (Bacci et al., 2015). In contrast, two different clones after bioassays were identified in two organic farms in Michigan (Dubey et al., 2012). The hypothesis of local epidemiology, whatever the circulating genotype, is also supported in our study by the heterogeneous distribution of seropositive animals according to the village. Of note, no swine clustered with the Africa 1 (DJO) strain, which has been reported to be more virulent in mice (Mercier et al., 2010). The minisequencing method, nevertheless, presents some limitations. The number of genetic markers (n = 8) is lower than the system based on 13 microsatellite markers (Mercier et al., 2010). The SNPs are also all in the same locus, the B1 gene—in contrast to the microsatellites markers which are more evenly spread across the genome. Studying one locus can introduce bias usually resulting in an underestimation of genetic differences. Therefore, a strict comparison with the genotypes reported in Africa using microsatellite markers (Mercier et al., 2010) was not possible. The main advantage of our system over microsatellite markers is the avoidance of a bioassay designed to amplify live parasites by inoculation in mice. This bioassay introduces bias when all animals from a given species are not all positive (Dubey et al., 2012; Mercier et al., 2010) or when T. gondii from a given intermediate host are not viable in mice (Aroussi et al., 2015). To understand the role of free-range pigs in the epidemiology of local toxoplasmosis and more specifically for human infection, more information is needed about the genotypes infecting the local populations. The seropositivity of animals strongly correlates with live, infective tissue cysts in meat (Dubey, 2009). Therefore, seropositive pork could be the source of human infection either directly, although this meat is either not consumed for religious beliefs or always consumed very well cooked in Burkina Faso, or indirectly if raw meat is consumed by feral cats in the vicinity of the slaughterhouse. The other method of human infection is indeed the ingestion of oocysts excreted by cats and the role of other intermediate hosts cannot be excluded. Poor sanitation could be a cause of contamination of the food by oocysts although oocysts are known to be rapidly killed at temperatures N 45 °C (Dubey, 1998)—this is often reached in summer months in Burkina Faso. Incidentally, if ingestion of environmental oocysts does not occur during the summer, the pigs are not progressively infected with increasing age and this can explain, at least in part, the lack of correlation between Toxoplasma seroprevalence in pigs and age. In addition, freeranging pigs live adjacent with human beings and are a sentinel of infection for the human population. The high prevalence of infection in swine reported here echoes what is seen in humans. Indeed, whatever the source of human infection, the Toxoplasma-seroprevalence in pregnant women is 33.1% at Bobo-Dioulasso. This suggests that maternal transmission to the fetus is possible (Bamba et al., 2014).
5. Conclusion The high rate of Toxoplasma seropositivity in Burkina Fasan freerange pigs highlights the need to monitor the spread of the parasite and the need to reduce its transmission. In addition, our study revealed variability of the genotypes circulating in small geographic areas, underlining the need for further genotype studies for epidemiological purposes. Genotyping of human cases in Burkina should help elucidate the source of infection.
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