Genotyping of T. gondii isolated from animal samples

Genotyping was performed with T. gondii-DNA isolated from oocysts or tachyzoites (if cultivation in VERO cells after bioassay in mice had been success-ful) utilising eight unlinked, independent chromosomal marker regions (newSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358 and PK1) and one extra-chromosomal marker region (Apico). An example of a PCR-RFLP analysis using the nine markers is shown in Figure 9.

A total of 104 T. gondii-positive isolates from Germany were examined.

Twenty-two isolates (collected between October 2004 and November 2006) had been only partially characterised in a previous study [Schares et al., 2008b]. All these isolates were now further analysed for the loci c22-8, c29-2, L358, PK1 and Apico [Su et al., 2006]. Oocysts of four isolates which could not be genotyped in the previous study because PCR amplification of the marker regions had failed were now fully typed by PCR-RFLP. The remaining isolates were analysed and genotyped for the first time. The typing results for the feline T. gondii isolates at all nine loci are summarised in Table 17. The majority (89/104; 85.58%) of these feline T. gondii isolates (collected October 2004–November 2006 and June 2007 – December 2010) displayed predominantly type II alleles. Of the 89 samples geno-typed as type II at all chromosomal loci, 83 possessed the type I allele (83/104;

79.81%) and six (6/104; 5.77%) displayed the type II allele at the Apico locus (Figure 10). One of the 104 isolates, TG-GER13, which could previously not be characterised, was shown to be of type III at all loci (1/104; 0.96%). No clonal type I isolate was detected. Among the 104 isolates, there were four isolates of non-canonical or mixed genotype (4/104; 3.85%). TG-GER02, which had previ-ously been characterised as type II at loci 5’ and 3’SAG2, BTUB, SAG3 and GRA6 only, was now shown to contain alleles of types I and II at loci c29-1 and PK1. TG-GER02 was thus identified as a non-canonical, mixed T. gondii isolate.

TG-GER20, which had not been typed previously, was of type II, except for the

loci Apico and BTUB. This isolate was characterised as type I at the Apico locus, but as types I or II at the BTUB locus. When PCR-RFLP were conducted for BTUB I and BTUB II separately, the isolate revealed the type II allele at the BTUB II locus, but type I and II or III alleles at the locus BTUB I. Isolate TG-GER61 showed a mixed allele pattern only at the Apico locus. Interestingly, iso-late TG-GER63 showed a mixture of type II and III alleles at some loci. While only type II patterns could be observed at the loci SAG3 and BTUB, type III al-leles were found at GRA6, L358 and Apico loci and a mixture of type II and III alleles at the c22-8, c29-2 and PK1 loci. In addition, a mixture of type I or III and II alleles was found at the newSAG2 locus in the isolate TG-GER63. Ten isolates (10/104; 9.62%) could not be fully genotyped at all nine loci (TG-GER09, 17, 34, 46, 51, 62, 83, 87, 100 and 104). TG-GER09 could only be genotyped at the loci newSAG2, GRA6, c22-8, L358 and Apico. All loci showed alleles of type II. TG-GER17 had alleles of type II at the loci newSAG2, SAG3, BTUB and GRA6, but showed a type I allele at the Apico locus. Isolate TG-GER34 could be genotyped at the loci SAG3, GRA6 and Apico and showed type II alleles at these loci. Iso-lates TG-GER46 and 52 showed alleles of type II at the loci BTUB, c22-8, L358 and PK1. Additionally, TG-GER51 had a type I allele at the Apico locus. Isolate TG-GER62 shared a type I allele at the Apico locus and a type II alleles at the newSAG2, SAG3, GRA6 and c22-8 loci. In isolate TG-GER83, a type I allele was observed at the Apico locus and a type II allele at the SAG3 locus. The loci c22-8 and c29-2 of TG-GER87 were of type II but the Apico locus showed a type I al-lele. Alleles at loci newSAG2, BTUB, c22-8, PK1 of isolate TG-GER100 were of type II, but the Apico locus showed a type I allele. No locus could be PCR-amplified from the TG-GER104 isolate. All results are summarised in Table 17 and Table 18.

Figure 9: PCR-PFLP polymorphism analyses of the T. gondii isolate TG-GER63.

Nine genetic markers (newSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) were utilised. All DNA fragments resulting from specific PCR and DNA endonuclease digestions were resolved in 3% agarose gels. Marker regions: newSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico; M: 100 bp DNA molecular size marker (Invitrogen, Germany); I, III, III:

types I (RH), II (ME49) and III (NED); lanes 1–4: T. gondii samples.

Figure 10: Proportions of differentT. gondii genotypes observed in oocysts shed by cats from Germany between June 2007 and December 2008.

Table 17: Multilocus genotyping ofT. gondii isolates from cats from Germany by PCR-RFLP analysis. nd: no amplification detected; a : previously genotyped using markers 3’ and 5’SAG2, BTUB, SAG3 and GRA6 only;* : DNA from tachyzoites after bioassay in GKO mice PCR-RFLP genotype (genetic marker) Apico I II III I II II II III

PK1 I II III II II I+II nd III

L358 I II III II II II II III

c29-2

I II III II II I+II nd III

c22-8

I II III II II II II III

GRA6 I II III II II II II III

BTUB I II III II II II nd III

SAG3 I II III II II II nd III

newSAG2 I II III II II II II III

T. gondii isolate RH ME49 NED 83 (TG-GER03a , 04a , 05a , 06, 08a , 10a , 11a , 12a , 14a , 15a , 16a , 18a , 19a , 21a , 22a , 23a , 24*, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 48, 49*, 52, 53, 54, 55, 56, 57, 58, 59, 60, 64, 65, 66, 67, 69, 71, 72, 73, 74, 75, 77, 79, 80, 81, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 105, 106) 6 (TG-GER07a , 50, 68, 76, 78, 85) TG-GER02a TG-GER09a TG-GER13a

Table 17 (continued)nd: no amplification detected; a : previously genotyped using markers 3’ and 5’SAG2, BTUB, SAG3 and GRA6 only;b : when analysed separately: BTUB I: type I and II or III allele; BTUB II: type II allele; * : DNA from tachyzoites after bioassay in GKO mice Apico nd I nd nd I I+II I III I I I nd

PK1 nd II nd II nd II nd II+III nd nd II nd

L358 nd II nd II nd nd nd III nd nd nd nd

c29-2 nd II nd nd nd II nd II+III nd II nd nd

c22-8 nd II nd II nd II II II+III nd II II nd

GRA6 II II II nd nd nd II III nd nd nd nd

BTUB II I+II b nd II II nd nd II nd nd II nd

SAG3 II II II nd nd nd II II II nd nd nd

newSAG2 II II nd nd nd nd II I or III+II nd nd II nd

T. gondii isolate TG-GER17a TG-GER20a TG-GER34 TG-GER46 TG-GER51 TG-GER61 * TG-GER62 * TG-GER63 TG-GER83 TG-GER87 TG-GER100 TG-GER104