Sequence Analysis of SAG2 of Feline Toxoplasma gondii Oocysts in Pakistan

Habibun Nabi1, Saher Islam2, Amna Arshad Bajwa2, Imran Rashid1,*, Haroon Akbar1, Wasim Shehzad2, Kamran Ashraf1, Nisar Ahmad1 and Aneela Durrani3

1Molecular Parasitology Laboratory Department of Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan

2Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan

3Department of Clinical Medicine and Surgery, University of Veterinary and Animal Sciences, Lahore, Pakistan

ABSTRACT

Toxoplasmosis is caused by coccidian parasite, Toxoplasma gondii. One third of human population of the world is believed to be infected with T. gondii. Cats serve as final host of Toxoplasma gondii and are the main source of contamination of soil and water. Fecal samples from cats at Pet center of UVAS (Lahore, Pakistan) were screened for coccidian parasites through microscopy examination. DNA was extracted from positive fecal samples for coccidian parasites and a T. gondii PCR was performed. Five sets of primers were designed using PrimerSelect tool for PCR to amplify SAG2 gene 5’ and 3’ regions. Sequences of 5 fragments of SAG2 were annotated and analyzed using DNASTAR Lasergene. After phylogenetic analysis with 3 clonal types and atypical strains, and on the basis of restriction map of HhaI and Sau3AI, our 3 isolates of T. gondii were found more closely linked to a typical strain. This is the first genetic analysis of T. gondii in Pakistan. In order to develop our knowledge about the toxoplasmosis epidemiology, further genotype analyses of T. gondii from animals and man need to be performed in Pakistan.

Article Information

Received 28 August 2016

Revised 14 March 2017

Accepted 23 May 2017

Available online 31 October 2017

Authors’ Contribution

IR conceived and designed the study. HuN, SI and ARB acquired, analyzed and interpreted the data. IR wrote the article. All others helped in preparation of manuscript.

Key words

Toxoplasma gondii, SAG2, Cat, PCR, Pakistan.

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.6.2067.2077

* Corresponding author: [email protected]

0030-9923/2017/0006-2067 $ 9.00/0

Copyright 2017 Zoological Society of Pakistan

Introduction

 

Toxoplasmosis is caused by an intracellular protozoan parasite, Toxoplasma gondii that may result in life-long colonization in animals and humans (Ali et al., 2017; Nicolle and Manceaux; 1909). This protozoa has cosmopolitan distribution and it is the most common human zoonotic infection in many geographic regions of the world (Scott et al., 2007). The infection can be acquired by three primary routes: ingestion of tissue cysts in undercooked infected meat; ingestion of food or water contaminated with sporulated oocysts shed in the feces of a cat; and congenitally, across the placenta from the mother to the fetus when she is infected through one of the previous two routes during pregnancy (Remington et al., 1985; Dubey, 1994). Please mention Remington before Dubey People may acquire infection by accidental intake of oocysts present in environment (soil, water, vegetables and fruits) contaminated with feces defecated by infected felines (definite host) (Benenson et al., 1982; Coutinho et al., 1982; Dubey et al., 2007; Baldursson and Karanis, 2011; Karanis et al., 2013). The role of cats in the contamination of environment with their fecal oocysts has been emphasized by health professional. Feral and owned cats contribute to spread T. gondii to humans and animals, as well as maintaining wildlife reservoirs (Frenkel et al., 1995; Weigel et al., 1995; Lehmann et al., 2003). A wide variability of fecal oocysts varying from 3 to 810 million, are shed by cats during 3 to 5 days after initial infection with T. gondii, and the shedding period lasts for a median of 8 days, although it may be as long as 3 weeks (Dubey, 1976, 2001, 2002, 2005). Oocysts may survive for months in soil and water, thereby enhancing the probability of transmission to intermediate hosts such as birds, rodents and humans (Yilmaz and Hopkins, 1972; Frenkel et al., 1975).

The genotyping studies on T. gondii’s led to the description of a clonal population structure with three main lineages, designated as type I, II and III, related to mouse-virulence (Darde et al., 1988, 1992; Howe and Sibley, 1995; Sibley and Boothroyd, 1992). Please rank the references Genotypes not belonging to the three main lineages were found predominant in Brazil where the population structure of Toxoplasma was more complex, with a higher genetic diversity than initially described. These “new” genotypes, or more exactly newly discovered genotypes, were designated, depending on the authors, as atypical, exotic, recombinant, or non-archetypal genotypes (Darde, 2008).

Several markers like SAG2, SAG3, PK1, BTUB, APICO, GRA6, c22-8, c29-2 and L358 have been used for T. gondii genotyping (Darde, 2004; Boughattas et al., 2014). However, the Surface Antigen 2 (SAG2) marker has been extensively used for strain identification (Sibley and Boothroyd, 1992; Parmley et al., 1994; Howe and Sibley, 1995; Howe et al., 1997; Mondragon et al., 1998; Owen and Trees, 1999; da Silva et al., 2005; Dubey et al., 2005a, b, 2006) into three clonal lineages and atypical strains (Behzadi et al., 2003; Wang et al., 2013). A preliminary study of the SAG2 gene, using restriction fragment length polymorphism (RFLP) method with HhaI restriction enzyme (Sibley and Boothroyd, 1992), found two alleles at this locus, including virulent specific and avirulent specific. Later, researchers developed the SAG2-RFLP marker using two enzymes, HhaI and Sau3AI, in PCR-RFLP that could distinguish the three major genotypes I, II and III (Howe et al., 1997).

We have collected fecal samples from cats and identified T. gondii using non-invasive method (Dabritz et al., 2007; Wendte et al., 2011). To the best of our knowledge, sequence analysis of SAG2 of T. gondii fecal oocyst was the pioneer study in Pakistan.

 

Materials and Methods

Fecal samples screening

Fecal samples from diarrheal cats were collected at Pet center, University of Veterinary and Animal Sciences (UVAS), Lahore, Punjab, Pakistan. The samples were screened microscopically for T. gondii on the same day and preserved in 70% ethanol at -20°C for further analysis.

Oocysts were used for DNA extraction using AxyPrep™ Multisource Genomic DNA mini-Prep kit following manufacturer instructions. Reference DNAs (RH) of Toxoplasma were used as positive samples. Five sets of primers were designed by PrimerSelect program (Wan and Fang, 2003) of DNASTAR Lasergene (Ahern, 1993; Burland, 1999) (DNAstar, Madison, WI) and used to amplify 5’ and 3’ regions of SAG2 gene (Table I). SAG2 gene was amplified from 5’ and 3’ end by performing polymerase chain reaction (PCR). Amplification was performed in thermo-cycler in a final volume of 20 μl of reaction mixture consisting of 2 μl of each primer (1 pM each), template (100 ng/μl), 10x PCR buffer with MgCl2 (25 mM), dNTPs (2 mM each) and 1 μl of Taq (5 U/μl) polymerase. The protocol for temperature cycling included 5 min at 94°C for initial denaturation, 35 cycles (45 sec of denaturation at 94°C, 1 min at annealing temperature of 64.6°C and 1 min of extension at 72°C). The final extension continued for an additional 10 min. The 3’ locus of SAG2 gene was similarly amplified by this standard PCR with specific primers. Products were electrophoresed in 1.2% agarose gel.

 

Table I.- Five sets of primer pairs for amplification of 5’ and 3’ regions of Toxoplasma gondii SAG2 gene.

Locus	Forward (F) and reverse (R) primers used
5’ Locus_171	F: 5'-AGTGACCCATCTGCGAAGAA-3' R: 5’-TTCTCAAAGACCACGAGCCT-3’
5’ Locus_242	F: 5'-TTCTCAAAGACCACGAGCCT-3' R: 5’-TGCACAGACTCGAGGAAGTT-3’
5’ Locus_211	F: 5'-CAGTGGCGAAGGTGATGTCT-3' R: 5’-CTCTCACGGGCAAGGTTCTT-3’
3’ Locus_288	F: 5'-CTCTCACGGGCAAGGTTCTT-3' R: 5’-CGAAGTTGGTGGTAACGGGA-3’
3’ Locus_230	F: 5'-CGCAGTTCTGTTCTCCGAAG-3' R: 5’-AGGAACTTGTTTGCCGACAC-3’

 

Genetic analysis

Before PCR products sequencing, amplified DNA were purified using ethanol precipitation. Briefly, 80% ethanol was added to PCR products and kept in dark for 30 min, followed by centrifugation at 12,000 rpm for 15 min. Pelleted material was air-dried overnight and diluted in distilled water.

The sequencing results were subjected to blast using Basic Local Alignment Search Tool (BLAST) provided by NCBI (McGinnis and Madden, 2004). Amplified DNA samples were sequenced by Sanger sequencing method at 1st BASE DNA Sequencing Services (Singapore) (Xie et al., 2004).

Sequenced fragments of SAG2 were annotated. Restriction enzyme analysis for HhaI and Sau3AI was carried out by using Sequence Builder program (Morales et al., 2012) of DNASTAR Lasergene. Alignment of sequences were performed using CLUSTAL W (Thompson et al., 1994) to quantify genetic distances among isolates of Type I, II, III and atypical ones found in Genbank as shown in Table II. We used MegAlign program (Fukushima et al., 2002) of DNASTAR Lasergene for phylogenetic tree construction and Bootstrapping analysis. The P (uncorrected distance often referred as p-distances or dissimilarity distance) and Jukes–Cantor distances were used to construct a neighbor-joining tree. We applied parsimony method with bootstrapping (1000 replicates) with MegaAlign program. SAG2 gene sequence of Neospora caninum was used as out-group (Ajzenberg et al., 2004).

 

Table II.- List of published SAG2 sequences along with their accession numbers.

Genotype	Accession number	Reference	Strain name
Type I	AK317818.1 M33572.1 JX045478 EU053942.1 EU258520	(Wakaguri et al., 2008) (Prince et al., 1990) (Khan et al., 2009) (Ferreira et al., 2008) (Dubey et al., 2008b)	RH RH RH RH RH
Type II	AF249697 AF357578 JX045473 JX045474 AB667974 EF585695 EF585696 EU053943.1 EU258521 EU258523 KC928258 KM246841 KM246837 KJ754425.1 KJ754389.1 KJ754409.1	(Lehmann et al., 2000) (Fazaeli and Ebrahimzadeh, 2007) (Khan et al., 2009) (Khan et al., 2009) (Tavalla et al., 2013) (Lindstrom et al., 2008) (Lindstrom et al., 2008) (Ferreira et al., 2008) (Dubey et al., 2008b) (Dubey et al., 2008b) (Burrells et al., 2013) (Donahoe et al., 2014) (Donahoe et al., 2014) (Vilares et al., 2014) (Vilares et al., 2014) (Vilares et al., 2014)	Beverley LGE96-1 DEG ME49 Tehran TgUgCh2 TgUgCh52 ME49 PTG TgWtdUs4 Pc10 NZfs8825 NZfs8825 G2992316 P1509306 P454205
Type III	AF249698 AF357577 AF357579 AB667973 AB667972.1 AB667975 DQ000461 EF585703 EU053944.1 EU258522 EU258528 KJ754400.1 KJ754396.1	(Lehmann et al., 2000) (Fazaeli and Ebrahimzadeh, 2007) (Fazaeli and Ebrahimzadeh, 2007) (Tavalla et al., 2013) (Tavalla et al., 2013) (Tavalla et al., 2013) (Sreekumar et al., 2005) (Lindstrom et al., 2008) (Ferreira et al., 2008) (Dubey et al., 2008b) (Dubey et al., 2008b) (Vilares et al., 2014) (Vilares et al., 2014)	C56 S48 NED S4 S7 S5 Skunk SAG2 TgUgCh52 VEG CTG TgWtdUs13 P3884739 P3216020
Atypical	AF249696 AF357582 AF357580 AF357581 JX045494 JX045491 JX045492 JX045493 JX045489 JX045472 JX045470 JX045471 EU258519 EU258531 EU258533 EU650329 EU650330 JX045490	(Lehmann et al., 2000) (Fazaeli and Ebrahimzadeh, 2007) (Fazaeli and Ebrahimzadeh, 2007) (Fazaeli and Ebrahimzadeh, 2007) (Khan et al., 2009) (Khan et al., 2009) (Khan et al., 2009) (Khan et al., 2009) (Khan et al., 2009) (Khan et al., 2009) (Khan et al., 2009) (Khan et al., 2009) (Dubey et al., 2008b) (Dubey et al., 2008b) (Dubey et al., 2008b) (Velmurugan et al., 2008) (Velmurugan et al., 2008) (Khan et al., 2009)	COUGAR TC751G34 CASTELLS MAS RUB COUG GUYKOE RUB GUYDOS GUYMAT CASTELLS TgCatBr1 MAS TgCgCa1 MAS TgDgCo11 TgCkGh1 TgCkNg1 VAND

 

Results

Sequence analysis of T. gondii

Five regions were amplified at 5’ and 3’ loci of SAG2 gene as shown in Figure 1. Five fragments of SAG2 were purified and got sequenced and were annotated. Clustal W analysis and then bootstraping were performed to find genotype comparison of our 3 laboratory isolates from cat fecal samples with published strains of Toxoplasma in GenBank. The 3 isolates of Toxoplasma were found to be more close to atypical strain AF357581 (Fig. 2). Three representative strains of each genotype from type I, II or III was taken after Clustal W analysis. The alignment of our 3 isolates was done with SAG2 sequences from GenBank of SAG2 T. gondii Type I, II and III strains at 5’. Dissimilarities among sequences after comparison at some points were observed (Fig. 3). Thus, it was found that our 3 isolates were near to atypical. Restriction enzyme analyses of Hha1 and Sau3AI were done by using Sequence Builder tool of DNASTAR Lasergene at 5’ and 3’ loci of SAG2 of our 3 isolates with representative isolates of Type I, II and III strains of T. gondii (Table III). Dissimilar pattern of restriction enzymes; Hha1 and Sau3A1 sites were observed in our isolates at 5’ and 3’ loci of SAG2 gene with the representative T. gondii strains of Type I, II and III.

 

Discussion

 

This study reveals new information on T. gondii genotype shedding from cat feces in the metropolitan city of Lahore, Pakistan. We confirmed the presence of T. gondii oocysts in cats’ feces through PCR, genetically characterized them at SAG2 locus and found diversity for atypical strain.

SAG2 polymorphic gene has been extensively used solely to characterize Toxoplasma genotype into 3 archetypal types (I, II and III) (Owen and Trees, 1999; Honore et al., 2000; Gallego et al., 2006; Abdel-Hameed and Hassanein, 2008; Asgari et al., 2013; Tavalla et al., 2013; Elamin, 2014) and atypical or recombinant types (Gallego et al., 2006; Lindstrom et al., 2006; Elamin, 2014).

 

We designed 5 primer sets to amplify 5’ and 3’ loci of SAG2 to see the polymorphic changes in the loci. Similarly, the researchers adopted this strategy to amplify separately SAG25’ and 3’ loci for genotyping analysis of Toxoplasma from clinical samples of patients and tissue samples of mice in Iran (Fuentes et al., 2001; Behzadi et al., 2003; Fallah et al., 2013).

Previously, most of the studies for genotyping analysis were done through ingestion of oocysts by rodents or from tissue cysts in mammals (Araujo et al., 2010; Cabral et al., 2013; Yan et al., 2014). All the tested fecal samples gave correct genotypes at least once for each locus when referenced against blood-derived genotypes (Lathuilliere et al., 2001).

 

Table III.- Restriction enzyme analysis of Hha1 and SauA3I. Analysis of restriction enzymes at 5’ SAG2 locus (A) and 3’ SAG2 locus (B).

A.- At 5’SAG2 locus.

Toxoplasma strains	HhaI position
	11	12	61	83	167	168
EU053942 (RH)	I	-	-	-	-	I
EU053943 (Me49)	-	II	-	-	-	II
EU053944 (VEG)	-	-	-	-	III	-
UVAS-Toxo-3	-	-	-	-	-	-
UVAS-Toxo-1	-	-	UVAS-Toxo-1	UVAS-Toxo-1	-	-
UVAS-Toxo-6	-	-	UVAS-Toxo-6	UVAS-Toxo-6	-	-

 

B.- At 3’SAG2 locus.

Toxoplasma strains	3 locus
	Sau3A1														Hhal
	29	58	63	73	107	152	164	165	173	186	198	199	207		120
AY895019 (RH)	-	I	-	I	I	-	-	-	-	-	-	-	-		-
EF585695 (TgUgCh2)	II	-	II	-	-	-	-	-	-	-	-	-	-		II
AB667975 (S5)	-	-	-	-	-	III	-	-	-	III	-	-	-		-
UVAS-Toxo-3	-	-	-	-	-	-	-	UVA S-To xo-3	-	-	-	UVA S-To xo-3	-		-
UVAS-Toxo-1	-	-	-	-	-	-	-	-	UVA S-To xo-1	-	-	-	UVA S-To xo-1		-
UVAS-Toxo-6	-	-	-	-	-	-	UVA S-To xo-6	-	-	-	UVA S-To xo-6	-	-		-

 

No study in Pakistan has been carried out to identify the genotype of Toxoplasma strain. The attempt to amplify SAG2 gene was unsuccessful by using the primers described by Howe and Sibley (1995) to amplify 1196 bp product for genotyping analysis. It might be due to the large size of fragment to be amplified. Experiencing the difficulties to optimize PCR on the published primers, we designed 5 sets of primers (Table I) to amplify 5’ and 3’ loci separately and contiguous sequence was constructed for alignment analysis with SAG2 published sequences of archetypal clonal and non-archetypal lineages. The 3 isolates from cat feces in Lahore metropolitan city were atypical. Vaudaux et al. (2010) showed that atypical strains possessed dissimilar sequences due to polymorphism in GRA6 and GRA7 genes that encoded different epitopes identified in Brazilian Toxoplasma isolates in chickens and cats from Santa Isabel (Vaudaux et al., 2010).

Different studies showed that the T. gondii population structure consists of three major clonal lineages designated types I, II, and III which are predominant in Europe and North America (Darde et al., 1992; Howe and Sibley, 1995), whereas atypical genotype is dominant in South America and Asia (Lehmann et al., 2006; Dubey et al., 2007, 2008a), some of which were also shown to be highly virulent in mice (Pena et al., 2008). T. gondii atypical genotypes were found to be associated with a number of severe cases of toxoplasmosis in immunocompetent individuals (Carme et al., 2009). Experimental studies showed that such atypical genotypes can develop when a cat ingests prey infected with T. gondii of more than one clonal Type, followed by sexual recombination in the gut of the cat which can result in progeny representing a mixture of the two parental genotypes (Su et al., 2002; Saeij et al., 2006). It is most likely that these atypical genotypes are the result of sexual recombination in cats (Herrmann et al., 2010). It is obviously similar to the prevalence of atypical strains in different ecological and geographical regions like South America.

Hha1 and Sau3AI were used for PCR-RFLP analyses at SAG2 loci to interpret genotyping studies of Toxoplasma (Howe et al., 1997; Fuentes et al., 2001; Grigg et al., 2001; Behzadi et al., 2003; Pena et al., 2006; Sabaj et al., 2010; Lass et al., 2012). We used Sequence Builder and MegAlign tools of DNASTAR Lasergene to characterize the restriction sites of Hha1 and Sau3AI of our isolates comparing with the representative T. gondii strains of 3 archetypical clonal lineages. Our isolates were atypical strains since dissimilar distribution pattern of restriction enzyme sites of Hha1 and Sau3AI of our isolates (Table III). These results are in comparison with Pena et al. (2006), who found 2.1% of mixed or recombinant T. gondii strains from tissue homogenates of 47 cats. They found dissimilar pattern of restriction digestion of Hha1 and Sau3AI of SAG2 locus as compared to Type I, II and III (Pena et al., 2006). Further studies are need to assess the genotype studies of T. gondii in intermediate hosts in humans and animals in Pakistan.

 

Acknowledgements

 

This study was funded by Grand Challenges Canada (Grant # S4_0266-01).

 

Statement of conflict of interest

Authors have declared no conflict of interest.

 

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