PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds

Blastocystis hominis was first reported as a yeast in human faecal samples in 1912 (Brumpt, 1912), and now this organism is accepted as a common protozoan parasite of the human intestinal tract (Windsor et al., 2002). The species name B. hominis is generally used for human isolates, while many B. hominis-like organisms have been isolated from various animals, especially from many species of mammals and birds (Abe et al., 2002; Burden et al., 1978/1979; Chen et al., 1997; Pakandl, 1991; Quílez et al., 1995). These isolates have been classified as B. hominis, Blastocystis sp. or newly proposed species, because there was no consensus for species designation of newly isolated Blastocystis from non-human hosts. So far, six and five isolates from different mammalian and avian species, respectively, have been proposed as new Blastocystis species, based on the differences in host origin, morphology, in vitro culture characteristics and/or karyotype (Belova, 1991, 1995; Belova & Kostenko, 1990; Chen et al., 1997; Krylov & Belova, 1997). However, Blastocystis is now recognized as a morphologically and genetically polymorphic organism and some non-human isolates have been demonstrated to be genotypically identical to B. hominis (Abe et al., 2003a, b, c; Arisue et al., 2003; Clark, 1997; Noël et al., 2003; Thathaisong et al., 2003; Yoshikawa et al., 1996, 1998, 2000, 2003, 2004). Therefore, if Blastocystis is a widely transmissible parasite between humans and mammals or birds, it is important to determine whether newly isolated non-human isolates are zoonotic. Recently we developed several sequence-tagged site (STS) primers derived from random amplified polymorphic DNAs (Yoshikawa et al., 1998, 2000, 2003). These primers can be used to identify the genotypes that correspond to phylogenetically different clades inferred from the small-subunit rRNA genes (SSU rDNA) (Arisue et al., 2003; Yoshikawa et al., 2003, 2004). Moreover, this PCR-based technique has recently been reported to be a practical tool for typing B. hominis isolates from humans and animals and for detecting some zoonotic genotypes from animal isolates (Abe et al., 2003a, b, c; Yoshikawa et al., 2003, 2004). In this study, Blastocystis isolates from a variety of mammals and birds, such as monkeys, cattle, pigs, chickens, quails and pheasants, were analysed with the STS primers and we demonstrate that most of the animal isolates were identical to B. hominis genotypes. In our previous studies, Blastocystis isolates were collected from various animals (Abe et al., 2002) and some isolates from monkeys, cattle, pigs and pheasants have been analysed with several STS primers (Abe et al., 2003a, b, c). However, some animal Blastocystis isolates were negative with STS primers that had allowed the classification of genotypes into subtypes 14. Therefore, in this study we examined these negative isolates and other isolates which have not been analysed previously. In total, 51 Blastocystis isolates from a wide range of animals were analysed by using PCR amplification with seven kinds of STS primers (Yoshikawa et al., 2003). The details of the isolates from different animals are shown in Table 1. The genomic DNA of Blastocystis organisms was extracted from cultures using DNAzol (Gibco-BRL) according to the manufacturer's protocol and then analysed using subtype-specific STS primer sets with PCR amplification to identify genotypes of B. hominis. Classification into subtypes 17 was examined, respectively, with primers SB83, SB155, SB227, SB332, SB340, SB336 and SB337, including a parallel control PCR amplification as described previously (Yoshikawa et al., 1998, 2000, 2003). The Nand II, B, HV93-13, HJ96AS-1, HJ96-1, SY94-3 and RN94-9 strains, which were sources of the STS primers, were used as a positive control for subtypes 17, respectively.

Table 1. Description of various Blastocystis isolates from mammals and birds The first two letters of the name of each isolate indicate the animal from which the organism was obtained, and the first number indicates the year of isolation. The number following the hyphen indicates the particular isolate in that year's series. The only exceptions are P1 and KS, which were isolated in Singapore. All isolates in the following series having the same prefixes (SY94, SY02, QQ93, QQ98 and QQ00) were obtained from the respective farms corresponding to the prefix. All other isolates were obtained from different farms located in different prefectures in Japan.

Since B. hominis populations isolated from humans and animals are extensively polymorphic in karyotype and SSU rDNA sequence (Arisue et al., 2003; Carbajal et al., 1997; Chen et al., 1997; Noël et al., 2003; Thathaisong et al., 2003; Upcroft et al., 1989), it is important to develop a tool to identify or classify different genotypes. We successfully developed PCR-based methodology for classification of different subtypes using STS primers developed from random amplified polymorphic DNAs (Yoshikawa et al., 1998, 2000, 2003). These amplified only distinct subtypes that corresponded to phylogenetically different clades inferred by SSU rDNA sequences (Arisue et al., 2003; Yoshikawa et al., 2003), so the STS primers could be used as a tool for typing of genotypes in Blastocystis populations.

When 51 isolates from various animals were screened with the seven kinds of STS primers, only 39 isolates were amplified by one of the primer sets, while four isolates were amplified by two primer sets and eight isolates were negative with all STS primers (Table 2). The isolates amplified by two STS primers were possibly mixed isolates containing two distinct genotypes, and the eight isolates (three from monkeys and five from pheasants) not amplified by any STS primers were probably unknown genotypes.

Table 2. Subtype classification of 51 isolates obtained from various mammals and birds

The results obtained in this study were combined with our previous studies on 41 isolates of animal origin (Abe et al., 2003a, b, c; Yoshikawa et al., 2003) and then compared with the distribution of human B. hominis subtypes obtained from five geographically different countries (Yoshikawa et al., 2004). Since subtypes 15 and 7 were observed in both human and animal isolates, these were possibly zoonotic genotypes (Table 3). Interestingly, the distribution of genotypes of animal isolates was different between mammalian and avian hosts. In the isolates from various mammals, subtypes 1, 3 and 6 were frequently observed. In contrast, subtypes 2 and 4 were the dominant genotypes in avian isolates, while subtypes 1 and 5 were rare except for all six isolates from chickens (Table 3). Although the five rodent isolates were isolated from different geographical origins (Japan, Singapore or United States), all these isolates were classified into subtype 7 (Yoshikawa et al., 2003) (Table 3). When variation of Blastocystis genotypes was compared between animal and human isolates, human isolates were more heterogeneous than animal isolates. Subtype 3 is the most popular genotype (60·8 %) in human isolates, while it is rare in animal isolates, for example 27·3 % in cattle and 8·8 % in pigs, suggesting that subtype 3 is of human origin. In contrast, subtype 6 is considered to be of animal origin because this genotype is only observed in the isolates from pigs (70·6 %) and cattle (54·5 %). In addition, all five rodent isolates were of subtype 7, but this subtype was rare in human isolates (3·9 %), suggesting that this genotype is also of animal origin. Although subtype 6 was not detected among 102 human isolates (Yoshikawa et al., 2004), this genotype branched between the clades including human B. hominis isolates (Arisue et al., 2003), suggesting that it may possess zoonotic potential. Therefore all of the animal isolates classified into the known genotypes appeared to be zoonotic isolates (Table 3). However, three isolates from monkeys and five isolates from pheasants were classified into unknown genotypes (Tables 2 and 3). We have analysed two human B. hominis isolates classified into unknown genotypes by phylogenetically inferring the full SSU rDNA sequence, and this showed that the two isolates were positioned into an additional clade supported by a 100 % bootstrap value (Yoshikawa et al., 2004). It is therefore important to survey B. hominis organisms to determine if new genotypes exist, and in conjunction with further studies on animal isolates, this will promote a better understanding of the genomic polymorphism of this parasite.

Table 3. Subtype classification with the STS primers among Blastocystis isolates from mammals and birds combined with our previous reports on animal and human isolates

We thank Dr M. Singh for providing us with genomic DNA of Blastocystis isolates P1 and KS. This study was supported by a grant from the Japan Society for the Promotion of Science to H. Y. (C-13670245).