Genetic diversity of Toxoplasma gondii isolates from Africa by PCR-RFLP and microsatellite genotyping: a systematic review

Faustina PAPPOE, WANG Lin, Dorcas OBIRI-YEBOAH, CHEN He, CHENG Wei-sheng,WANG Xiao-wei, LIU Fang, LUO Qing-li, SHEN Ji-long

(1. Department of Parasitology, Provincial Laboratory of Microbiology & Parasitology andthe Key Laboratory of Zoonoses Anhui, Anhui Medical University, Hefei 230032, China;>2. Department of Microbiology and Immunology, School of Medical Sciences, University of Cape Coast, Ghana;3. Clinical Laboratory, the First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230031, China;4. Clinical Laboratory, the First Affiliated Hospital of Anhui Medical University, Hefei 230032, China)



Genetic diversity of Toxoplasma gondii isolates from Africa by PCR-RFLP and microsatellite genotyping: a systematic review

Faustina PAPPOE1,2, WANG Lin3, Dorcas OBIRI-YEBOAH2, CHEN He4, CHENG Wei-sheng1,WANG Xiao-wei1, LIU Fang1, LUO Qing-li1, SHEN Ji-long1

(1.DepartmentofParasitology,ProvincialLaboratoryofMicrobiology&ParasitologyandtheKeyLaboratoryofZoonosesAnhui,AnhuiMedicalUniversity,Hefei230032,China;>2.DepartmentofMicrobiologyandImmunology,SchoolofMedicalSciences,UniversityofCapeCoast,Ghana;3.ClinicalLaboratory,theFirstAffiliatedHospitalofAnhuiUniversityofChineseMedicine,Hefei230031,China;4.ClinicalLaboratory,theFirstAffiliatedHospitalofAnhuiMedicalUniversity,Hefei230032,China)

The genetic diversity ofToxoplasmagondii(T.gondii) population varies from one geographical area to another. The majority ofT.gondiiisolates in North America and Europe are types II and III. In South America no clonal or atypical genotype is dominant. In Asia, the atypical ToxoDB# 9 and clonal type I are dominant. Very few studies onToxoplasmagenotypes have been done in Africa. The available information is from northern, western, eastern and central Africa. The aim of this study was to analyze and highlight the population genetic structure ofT.gondiiisolates circulating in Africa revealed by multi-locus PCR-RFLP and microsatellite typing. In this paper, published information on the population structure ofT.gondiiisolates from African humans and animals was reviewed. Articles were retrieved from Pubmed, Science Direct, Google Scholar data bases and ToxoDB. From 13 retrieved publications,Toxoplasmaisolates were identified in 17 humans, thus, atypical genotype (76.5%, 13 isolates predominant due to Africa 1, 69.2%, 9 isolates), type I (5.9%, 1 isolate) and mixed genotype (17.6%, 3 isolates). Of 1 660 domesticated animals 314T.gondiiisolates were recovered, thus, type II (42.7%, 134 isolates), type III (32.8%, 103), atypical genotype (19.4%, 61), type I (1.9%, 6) and mixed genotype (2.1% 10 mixed isolates). In total, the proportions of the genotypes were 40.5% (134/331), 31.1% (103/331), 22.3% (74/331), 2.1% (7/331) and 3.9% (13/331) for type II, type III, atypical genotype, type I, and mixed genotype respectively. Deductively, 73.7% of the isolates were archetypical. The available information indicates that, both archetypical and non-archetypical populations ofT.gondiioccur in Africa and infection caused by archetypical genotype ofT.gondiiis very high in both human and animal population from Africa.

Toxoplasmagondii; population structure; Africa; PCR-RFLP; microsatellite genotyping

Introduction

Toxoplasmosis is an important world-wide distributed zoonotic infection of most mammals including humans. It is caused by an obligatory intracellular protozoan,Toxoplasmagondii(T.gondii)[1-2]. Acquisition is mainly by ingesting raw and undercooked infected meat containing viableToxoplasmatissue cysts or food or drink contaminated withToxoplasmaoocysts excreted from the feces of infected cats, the only definitive host[1-2]. Blood transfusion and organ transplantation are also possible means of acquiring the infection in human. Congenital toxoplasmosis occurs through trans-placental transmission of tachyzoites from mother with active infection to the developing foetus[1-2].

Globally, it is estimated that approximately 30% of the human population is chronically infected. However, most immunocompetent individuals are asymptomatic during acute infection[1,3]. But mild to severe clinical manifestations including lymphadenopathy and neuropsychiatric symptoms such as, depression, anxiety, schizophrenia, and suicide have been reported in some so-called asyptomatic persons with acute or chronic infection[4-7]. It has been observed that infection in rats causes them to be fearless near cats and this increase the chance of being preyed upon by the cats[8]. Another undesirable consequence is thatT.gondiiinfection acquired during pregnancy may lead to congenital toxoplasmosis which may result in abortion, neonatal death and severe morbidity[9-11]. In immunocompromised individuals such as AIDs, cancer and organ transplantation patients, primary or reactivation of latent infection can cause life-threatening encephalitis or lethargy[12-14]. Additionally, infection in animals causes a considerable economic loss to the animal husbandry industry as a result of increased abortion, mortality and medical cost. Hence,T.gondiiinfection is of public and veterinary importance[15-16].

The variation in the clinical manifestations has led to researches on the genetic diversity and/or pathogenicity amongT.gondiiisolates from healthy and sick hosts from different geographical areas. Recent studies have revealed thatT.gondiihas a complex genetic structure than was initially recognized[17]. Isolates ofToxoplasmaare categorized into two groups through the use of multi-locus enzyme electrophoresis, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or microsatellite typing techniques[17-21]. The identified groups are clonal lineage also known as archetypical or typical strains which are distributed worldwide and atypical strains also known as exotic or non-archetypical strains[17-21]. The clonal lineage strains are predominant in North America and Europe[19]. They include type I, the highly virulent strain and types II and III, the intermediate or avirulent strains in mice[19]. Atypical strains constitute all other genotypes that do not fall into archetypical genotypes. These genotypes may be produced when the definitive host, felids are simultaneously infected with multiple strains[22]. Atypical strains also range from highly virulent to intermediate or avirulent phenotypes[22-25]. Atypical strains such as Africa 1 and Africa 3 have been isolated in Africa[26-27]. In Asia, and particularly in China, Chinese 1 has been reported as the predominant atypical genotype[28-30]. Several studies in Europe, North and South America have also revealed atypical strains and the atypical strain, type 12 is predominant in wild animals[31-33]. From the available literature it can be concluded that the genetic diversity ofT.gondiipopulation varies in geographical areas.

This paper presents a review of the genetic diversity ofT.gondiiisolates from Africa revealed by multi-locus PCR-RFLP and microsatellite typing.

MethodsLiterature search

A comprehensive literature search of the Pubmed, Science Direct, Google scholar data bases as well as ToxoDB was performed up to August, 2015. The following keywords were used: “Toxoplasmagondiigenotyping studies in Africa”. This was followed by another search using the keywords “toxoplasmosis in Africa”. Furthermore, a search was performed on the relevant references from the selected publications. EndNote X7 (Thomson Reuters) was used for literature management.

Data collection

All retrieved studies were reviewed carefully by two investigators, FP and LW. We included primary articles without sample size and study design restriction. All the studies included involved the analyses of the genetic structure ofT.gondiiisolates from human and/or animals using PCR-RFLP and /or microsatellite genotyping with five or more genetic marker. Studies that were only presented as abstract and those that recruited non-African and unspecified population were excluded. Also, we excluded studies that presented inconsistent results. Thus, we excluded any article in which the results in the main text were different from the results presented in tables or abstract.

Data analysis

Very few studies onToxoplasmagenotypes have been done in Africa although the available information is from northern, western, eastern and central Africa. No data, however, were reported from southern Africa (Figure 1). From the four databases (Pubmed, Science Direct, Google Scholar and ToxoDB), a total of 13 publications were considered eligible for our studies. This composed of four publications on human and nine on animals. Three of the studies used microsatellite genotyping method (Tables 1 and 2). The retrieved studies on human population involved 17 individuals including congenital and HIV/AIDS individuals.Toxoplasmaisolates were identified in all the 17 humans. Our analysis revealed TWO different genotypic groups, clonal type I (#10) and atypical genotype (Table 1). Mixed genotypes were also identified. Proportionally, the atypical genotype was found to be predominant accounting for 76.5% (13 isolates). Mixed genotype isolates were three accounting for 17.6% while clonal type 1 accounted for 5.9% (1 isolate). Out of the 13 individual atypical genotypic isolates, Africa 1 (I/III) was the most dominant accounting for 69.2% (9 isolates). Also, the only study which investigated the virulence of the isolate in mice reported on I/III isolate being virulent to mice (Table 1). On the other hand, in animals, a total of 314T.gondiiisolates were recovered from 1 660 domesticated animal samples. Our analysis showed that 134 isolates belonged to type II (42.7%), 103 to type III (32.8%), 61 to atypical genotype group (19.4%) and 6 to type I (1.9%) (Table 2). Mixed genotype isolates constituted the remaining 3.2% (10 mixed isolates). It was observed that, all the nine retrieved articles carried out parasite virulence in mice or cell culture. However, only six studies clearly stated the virulence status of the isolated strains (Table 2).

Bf: Burkina Faso; DROC: Democratic Republic of Congo; Eg: Egypt; Et: Ethiopia; Gb: Gabon; Gh: Ghana; Ke: Kenya; Ma: Mali; Ng: Nigeria; Ug: Uganda; Tu: Tunisia.Fig.1 Map of Africa showing the geographic origins of T. gondii isolates

In both human and animals, a total of 331 isolates were identified from 1 677 samples. The proportions are presented in Figure 2. Collectively, the clonal genotypes accounted for 73.7% (244/331): majority of the isolates being type II (54.9%; 134/244) and type III (42.2%, 103/244) whiles type 1 was the least occurring one (2.9%, 7/244). Atypical genotype group accounted for 22.3% (74/331). Mixed genotype isolates made up the remaining 3.9% (13).

Type I, 2.1% (7/331); Type II, 40.5% (134/331); Type III, 31.1% (103/331); Atypical, 22.3% (74/331); and mixed genotype infection, 3.9% (13/331).Fig.2 The total proportion of T.gondii isolates from the nine retrieved studies

Tab.1 Genotypes of T. gondii isolates from human population in Africa

Countryoforiginofpatients/infectionPopulationsurveyedSamplesizeConventionalgenotypedesignationTypeITypeIIclonalorvariantTypeIIIAtypicalMixedTotalNo.ofisolatesGenotypingmethodReferencesGhanaHIV/AIDS1I/III(1;*)1Microsatellite(fivemarkers)[34]TunisiaCongenitaltoxoplasmosis14I(1;*)I/III(7;*)I/II(3;*)I/II+I/III(3;*)14RFLP-PCR(sixmarkers)[26]TunisiaNeonate1I/III(1;V)1RFLP-PCR(sixmarkers)[35]TunisiaFetus1MixofI/II/III&atypicalalleles(1;*)1RFLP-PCR(Tenmarkers)[36]Total171(5.9%)——13(76.5%:I/III=69.2%)3(17.6%)17(100%)

Note： V means the strain was virulent in mice; ＊means virulence of strain not investigated. I/III= Africa1

Tab.2 Genotypes of T. gondii isolates from animal population

Countryoforiginofpatients/infectionHostSamplesizeConventionalgenotypedesignationTypeITypeIIclonalorvariantTypeIIIAtypicalMixedTotalno.ofisolatesGenotypingmethodReferenceGhanaChicken64Atypical(2;Av)2PCR-RFLP(Tenmarkers)[37]EgyptChicken7IIvariant(2;θ)III(5;θ)7PCR-RFLP(Tenmarkers)[21]MaliChicken4IIvariant(1;θ)III(3;θ)4BurkinaFasoChicken1III(1;θ)1DROCChicken5IIclonal(1;θ)III(4;θ)5KenyaChicken1IIvariant(1;θ)1NigeriaChicken79Atypical(1;θ)1UgandaChicken85I(6;Av)IIclonal(8;Av)III(1;Av)I+II(3;Av)I+II+III(2;Av)20PCR-RFLP(fivemarkers)[38]EgyptCat137IIclonal(59;θ)IIvariant(2;θ)III(42;θ)Atypical(8;θ)mixedgenotypeinfection(4;θ)115PCR-RFLP(Tenmarkers)[39]表2(续)Countryoforiginofpatients/infectionHostSamplesizeConventionalgenotypedesignationTypeITypeIIclonalorvariantTypeIIIAtypicalMixedTotalno.ofisolatesGenotypingmethodReferenceGabonCatChickenGoatSheep425III(35;Av)I/III(11;V)III-like(3;Av)Africa3(19;V)Singlegenotype(1;Av)69Microsatellite(13markers)[27]Ethiopiachicken125IIclonal(1;Av)1PCR-RFLP(Tenmarkers)[40]EgyptStraydogs51IIvariant(11;Av)III(4;Av)#20(6;Av)Mixedgenotypeinfection(1;AV)22PCR-RFLP(Tenmarkers)[41]EthiopiaCats48IIclonal(9;θ)IIvariant(10;θ)III(5;θ)#20(9;θ)33PCR-RFLP(Tenmarkers)[42]EthiopiaSheepGoat628IIclonal(29:1V;28Av)III(3:1V;2Av)Atypical(1;V)33Microsatellite(15markers)[43]

Note： V, Av and θ represent virulence, avirulence and virulence status not mentioned respectively.

Discussion

Toxoplasmagondiiinfections are prevalent in both humans and animals worldwide. It is known that the development, progression and severity of disease depend on the immunological status of the infected host. However, recent studies have showed that the genotype of the parasite is also a contributing factor[19,22-25]. Therefore understanding the genetic structure ofT.gondiipopulation is very crucial in providing information about the relationship between the genotypes and the associated phenotypes. Recent studies using multi-locus enzyme electrophoresis, PCR-RFLP or microsatellite typing to genetically characterizedT.gondiiisolates have showed that the population structure of this parasite is complex. In Asia, particularly in China and Vietnam, type I and Chinese 1 are the frequently isolated strains from both human and animals[28, 44-46]. In Europe and North America, the archetypical genotypes, types II and III are reported to be predominant[20,47-48]. The atypical strain, type 12 is also common in North America isolated mainly from wildlife[31-33,49]. However, the isolates from South America are more genetically diverse with no genotype being clearly dominant[50-52]. In Africa,T.gondiiisolates belonging to the clonal lineages, I, II and III have been frequently isolated in addition to Africa 1 and Africa 3. However, some researches from Africa relied on a single locus which is not appropriate to characterizeToxoplasmagenotype.

This study was conducted with the aim of analyzing and highlighting the population genetic structure ofT.gondiiisolates circulating in Africa revealed by multi-locus PCR-RFLP and microsatellite typing.

Our analysis indicates that, both typical and atypical population ofT.gondiioccur in Africa. The atypical genotype accounted for 76.5% of the human infection with Africa 1 (I/III) being the most dominant one (69.2%). Less than 20% of the infection was caused by mixed genotype (17.6%) and clonal type 1 was (5.9%). This pattern is not different from what is obtained in Asia mainly in China and Vietnam where atypical strain (Chinese 1) and type I have been frequently isolated from cancer patients[45-46]. However, types II and III which have been frequently reported in AIDS patients and congential infection from North America and Europe were not identified in this study[20,51-52]. With the exception of one study, all the studies involving human population did not investigate the virulence of the isolates in mice. This could be partly due to insufficient resources. However, most of the isolates obtained from animals were type II (42.7%) and type III (32.8%) which have also been frequently isolated from animals in North America and Europe. Atypical genotype group accounted for 19.4% and the least was type I (1.9%). Multiple genotype infection in the same animal hosts constituted the remaining 3.2%. Some of the strains were virulent whiles others were avirulent in mice or cell culture[27,37-38,40-41,43].

Overall, the archetypical genotype predominates in Africa accounting for 73.7%: with types II and III dominating indicating that, types II and III genotypes are not merely predominant in Europe and North American but also predominant in Africa. This is consistent with the study done by Shwab et al.[53]who reported the global pattern ofToxoplasmagondiigenetic diversity revealed by multilocus PCR-RFLP genotyping only.

Conclusion

Totally, infection caused by archetypical genotype ofT.gondiiis very high in both human and animal population from Africa. Our result will contribute to the information available on the control of this opportunistic pathogen that mainly cause severe disease among immunocompromised diseased individuals like AIDS patients and also in congenital cases. However, more sampling is required in Africa including southern African countries where no studies about the genetic structure ofT.gondiihave been reported. More studies on human toxoplasmosis are also very important to help to obtain representative isolates from the various geographical regions of the African continent. Also, it was observed that all the studies on animals were biased toward domesticated animals. More studies are needed to include wild animals for better understanding of the genetic structure ofT.gondiipopulation in Africa.

Competing interests

The authors have no competing interests

Authors’ contributions

JLS, FP and LW conceived and designed the study. JLS critically revised the manuscript. FP and LW performed database information search. FP wrote the manuscript. FP, LW, DOY, HC, CWS, XWW, FL and QLL participated in the analysis and interpretation of data. All authors have read and approved the final manuscript.

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Received:2016-04-21；Revision accepted:2016-07-22

国家基础科学研究项目(No. 2010CB530001)，国家自然科学基金(No.81471983)

1. 安徽医科大学病原生物学教研室，合肥 230032；

2. Department of Microbiology and Immunology, School of Medical Sciences, University of Cape Coast, Ghana；

3. 安徽中医学院第一附属医院检验中心，合肥 230031；

4. 安徽医科大学第一附属医院，合肥 230032

DOI：10.3969/j.issn.1002-2694.2016.010.012

刚地弓形虫非洲分离株的PCR-RFLP和微卫星基因分型研究进展

Faustina PAPPOE1,2,王 林3, Dorcas OBIRI-YEBOAH2,陈 鹤4, 程维晟1, 王晓玮1,刘 芳1, 罗庆礼1, 沈继龙1

刚地弓形虫(T. gondii)的遗传多样性与地理区域相关。北美和欧洲的弓形虫基因型以II型和III型为主。南美洲以非典型基因型为主。亚洲弓形虫的优势基因型主要为Chinese 1 型(ToxoDB# 9) 和I型。目前关于非洲弓形虫基因型的研究较少。本文对源于北非、西非、东非和中非地区弓形虫的多位点PCR-RFLP和微卫星分型以及非洲地区弓形虫群体遗传结构进行了综述。从17例患者中分离出弓形虫，非典型基因型13 株(76.5%), 主要为 Africa 1 9 株(69.2%)；I型1株(5.9%) 以及混合基因型3株(17.6%)。从1660饲养动物中分离出314株，其中II型134株(42.7%), III型103株(32.8%)，非典型基因型61株(19.4%), I 型6株(1.9%) 和混合基因型10株(3.2%)。人畜弓形虫基因型的比例为分别为：II 型40.5% (134/331), III 型31.1% (103/331)，非典型基因型22.3% (74/331)，I型2.1% (7/331) 和混合基因型3.9% (13/331)。典型基因型占73.7%。综上可知，非洲弓形虫基因型包括典型和非典型基因型，前者在非洲人群与动物中均有较高的感染率。

刚地弓形虫、种群结构、非洲、PCR-RFLP、微卫星基因分型

R382.5

A

1002-2694(2016)10-0903-08

10.3969/j.issn.1002-2694.2016.10.011

Shen Ji-long, Email: shenjilong53@126.com

Funded by the National Basic Science Research Program of China (Grant No. 2010CB530001) and the National Natural Science Foundation of China (Grant No. 81471983)