Molecular Survey of Enterocytozoon bieneusi in a Japanese Porcine Population

E nterocytozoon bieneusi was first detected in a HIV-positive patient in 1985, and has been the most frequently found microsporidian species in humans, particularly immunocompromised AIDS patients with chronic diarrhea (Mathis et al. 2005). Probable modes of transmission to humans include the fecal–oral route via ingestion of food and water contaminated with spores that are relatively resistant to the environment (Mathis et al. 2005). Although the precise source of infection in humans remains uncertain, a variety of animals, including domestic, wild, and companion animals, as well as infected humans, are considered to be the main source of infection, as genotypes that infect humans have been identified in these animals (Mathis et al. 2005). Epizootiological studies have confirmed that infection with E. bieneusi in pigs is more prevalent than in other animals such as cattle, dogs, cats, and wild animals (Mathis et al. 2005). Moreover, to the best of our knowledge, pigs harbor 2 zoonotic genotypes in addition to 14 animal-specific genotypes (13 of these 14 genotypes are pig specific) (Breitenmoser et al. 1999, Rinder et al. 2000, Dengjel et al. 2001, Buckholt et al. 2002). Therefore, genotyping of the isolates from animals, especially from pigs, may prove significant for the adequate control of E. bieneusi infections.

We recently showed for the first time the presence of E. bieneusi in dogs and a cat in Japan using polymerase chain reaction (PCR) and direct sequencing of the internal transcribed spacer (ITS) region of the rRNA gene (Abe et al. 2009). However, the prevalence of this pathogen in pigs maintained in Japan remains to be investigated. It appears likely that the prevalence of E. bieneusi infection is high in pigs raised in Japan. In the present study, we examined 30 fecal samples from pigs reared on six farms in western Japan using PCR and identified the genotypes using sequence analysis of the ITS region.

A total of 30 pigs that were approximately 6 months of age were randomly selected from six farms (A–F) in six different prefectures in western Japan (Table 1). Since these farms were geographically separated and were solely hog operations, and given that the breeder was only in contact with the animals reared on their farm, there was no possibility of contamination from cattle to pigs, or from farm to farm. The pigs were transferred to the meat inspection center of western Japan, where fecal samples were collected from the rectum in August 2008. At the time of collection, all animals appeared clinically healthy. All samples were collected in 50 mL sterilized plastic tubes using disposable spoons and stored at 4°C until examination. Purification of spores from fecal samples and extraction of genomic DNA were accomplished within 1 week of collection. Approximately 2 g of each fecal sample was thoroughly suspended in phosphate-buffered saline and centrifuged at 1200 g (2500 rpm) using the Parasep (Intersep, Novi, MI) to remove fecal debris. After centrifugation, the supernatant was discarded and the filtrate, including spores, was gently mixed with 500 μL of phosphate-buffered saline. Two hundred microliters of each specimen was then subjected to three rounds of freeze–thawing and boiled for 10 min. Genomic DNA was then extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The ITS and a portion of the large and small subunit ribosomal RNA gene regions were amplified using nested PCR and primers specific for E. bieneusi (Buckholt et al. 2002). The primers were termed EBITS3 and EBITS4 for the first PCR, and EBITS1 and EBITS2.4 for the second PCR. E. bieneusi genotype Type IV DNA originating from a cat (Abe et al. 2009) was used as a positive PCR control. Amplification products were purified using the QIAquick Gel Extraction Kit (Qiagen) or QIAquick PCR Purification Kit (Qiagen) and sequenced in both directions using the EBITS1 and EBITS2.4 primer pair on an ABI 3130 automated sequencer (Applied Biosystems, Foster City, CA). Sequence chromatograms from each strand were aligned and analyzed using the SEQUENCHER Version 4.1 (Gene Codes, Ann Arbor, MI). The obtained 243 nucleotide sequence of the ITS region was compared with sequences in the DNA database (GenBank/European Molecular Biology Laboratory, EMBL/DNA Data Bank of Japan, DDBJ) by FASTA analysis (DDBJ, http://fasta.ddbj.nig.ac.jp/top-j.html). Nucleotide sequence data obtained in the present study are available in DNA databases (GenBank/EMBL/DDBJ) under accession numbers AB470279 to AB470284. A consensus paper that reports E. bieneusi genotyping has been recently published (Santín and Fayer 2009). To reduce confusion associated with the identification of the genotypes within E. bieneusi, it was proposed that the first published name should have precedence and thus become the primary name used in all subsequent publications. In the present study, we followed this recommendation and used the primary genotype names.

A specific 390 bp fragment was successfully amplified in 10 DNA samples from pig (data not shown). The number of positive cases varied among farms; however, positive cases were found in all of the farms examined (Table 1). Although the number of samples examined was relatively small, the overall prevalence of E. bieneusi infection in the 30 pigs was calculated to be 33%. This prevalence in pigs was similar to the data obtained using PCR techniques in Switzerland (35%, 38/109) and the United States (32%, 64/202) (Breitenmoser et al. 1999, Buckholt et al. 2002). Given that the analyzed farms were randomly selected and that there was no possibility of cross contamination from farm to farm, the result obtained in the present study indicates that E. bieneusi is widespread on farms in Japan.

Sequences of the four isolates from farm B and the two isolates from farm F were unique. The homology of the ITS region identified using the FASTA program showed that the sequences of the isolates from farms A, B, C, E, and F were 100% identical to those of the E. bieneusi isolates belonging to the genotypes EbpA (syn. F, accession number AF076040), H (AF135835), PigEBITS5 (AF348473), D (syn. PigITS9, WL8, Peru9, PtEb VI, CEbC, AF101200), and EbpC (syn. E, WL13, WL17, Peru4, AF076042), respectively. Among these genotypes, EbpA has been identified in pigs and cattle from Switzerland and Germany (Breitenmoser et al. 1999, Rinder et al. 2000), while the genotypes H and PigEBITS5 have been identified in pigs in Germany and the United States (Rinder et al. 2000, Buckholt et al. 2002). Considering the previous epizootiological data, the result obtained in the present study strongly suggests that genotypes H and PigEBITS5 are pig specific and that the three genotypes EbpA, H, and PigEBITS5 are geographically widespread. In contrast to these host-specific genotypes, genotypes D and EbpC are considered zoonotic genotypes, as the same genotypes have also been found in humans (Sulaiman et al. 2003, Breton et al. 2007). In pigs, both genotypes have been reported in the United States (genotype D) and Switzerland (genotype EbpC) (Breitenmoser et al. 1999, Buckholt et al. 2002). The present study suggests that both zoonotic genotypes are also geographically widespread, and that pigs may play an important role as a zoonotic reservoir in Japan.

The sequence of one isolate from farm D revealed the potential presence of “mixed templates” characterized by two overlapping nucleotide (G and A) peaks at positions 31 and 136 from the 5′ terminus of the 243-bp nucleotide sequence. As a result, four sequences, G and A, G and G, A and G, and A and A, at positions 31 and 136, respectively, were predicted. According to homology searches, the sequence containing nucleotides G and A at positions 31 and 136, respectively, was 100% identical to that of genotype H (AF135835). Similarly, the sequence containing nucleotide A at both positions was also 100% identical to that of genotype EbpA (AF135833). The other predicted sequences containing nucleotide G at both positions (99.6% similar to genotype H) or nucleotides A and G at positions 31 and 136 (99.6% similar to genotype EbpA) did not correspond to any other sequences available in the international DNA databases (GenBank/DDBJ/EMBL), indicating new genotypes. Interestingly, similar overlapping nucleotide G and A peaks at the same positions mentioned above have also been found in isolates from pigs raised in Germany (EU849130, deposited by Broglia and Reetz 2008). This genotype had been registered as the mixed genotype H/EbpA; however, the sequence data have only been published in the databases. Therefore, it is likely that the two overlapping nucleotide peaks located at the specific positions found in the sequences of the isolate from farm D are due to mixed infection with different genotypes such as H, EbpA, and possible new genotypes.

E. bieneusi is recognized as a major etiological agent of chronic diarrhea in HIV-infected patients or in immunocompromised patients due to therapeutic immunosuppression when undergoing organ transplantation (Mathis et al. 2005). The prevalence of E. bieneusi infections among HIV-infected patients has increased by up to 50% of that previously reported (Mathis et al. 2005). Between 1985 and December 31, 2007, 10,864 HIV cases and 4468 AIDS patients were reported in Japan. Although the increase in reported HIV cases in Japan has accelerated and the spread of HIV infection has not ceased (Infectious Disease Surveillance Center 2008), no clinical cases of E. bieneusi infection in HIV-positive and AIDS patients have been reported in Japan. This is likely due to the fact that E. bieneusi may be overlooked at Japanese medical centers, as this pathogen is not known to Japanese physicians and medical technologists. Even though people working closely with animals may be at risk of becoming infected with E. bieneusi, investigation and identification of this pathogen in the human population remains to be undertaken. Therefore, further fecal examinations of a variety of animals, people who handle animals such as farmers, as well as patients with chronic diarrhea are required to precisely clarify the epidemiology of E. bieneusi infection in Japan.