• View in gallery

    Muscle biopsy tissue, compressed between glass slides, showing non-encapsulated Trichinella larvae.

  • View in gallery

    Multiple alignment of representative DNA sequence obtained from imported Trichinella Malaysian isolate (Trichinella_pa-ma). Nucleotide sequences from mitochondrial RNA genes of 11 Trichinella species and genotypes—Trichinella papuae, Trichinella zimbabwensis, Trichinella pseudospiralis, Trichinella spiralis, Trichinella nelsoni, Trichinella nativa, Trichinella sp. T6, Trichinella murrelli, Trichinella sp. T9, Trichinella britovi, and Trichinella sp. T8 (GenBank accession nos.: EF517130, EF517131, EF517124, GU339146, EF517127, DQ159093, EF517126, EF517125, EF517129, DQ159092, and EF517128, respectively)—were used for comparison. Dots represent sequence identity with Trichinella Malaysian isolate. Gaps are represented by dashed lines.

  • View in gallery

    Multiple alignment of partial DNA sequence of cytochrome c oxidase subunit I (COI) gene obtained from four isolates of Trichinella papuae. An imported Trichinella Malaysian isolate (Trichinella_pa-ma) was aligned with three isolates of T. papuae in GenBank (accession nos.: EF601546, DQ007899, and GQ180179). Dots represent the conservation of the base among all the examined isolates. Horizontal bars represent the position of polymerase chain reaction (PCR) primers.

  • 1.

    Dupouy-Camet J, Murell KD, 2007. FAO/WHO/OIE Guidelines for the Surveillance, Management, Prevention and Control of Trichinellosis. Paris: FAO/WHO/OIE, 1119.

    • Search Google Scholar
    • Export Citation
  • 2.

    Gottstein B, Pozio E, Nockler K, 2009. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol Rev 22: 127145.

  • 3.

    Boonthahom P, Nawarat A, 1963. The outbreak of trichinosis at Amphur Mae Sarieng. Bull Public Health 33: 301308.

  • 4.

    Khamboonruang C, 1991. The present status of trichinellosis in Thailand. Southeast Asian J Trop Med Public Health 22 (Suppl): 312325.

  • 5.

    Takahashi Y, Mingyuan L, Waikagul J, 2000. Epidemiology of trichinellosis in Asia and the Pacific Rim. Vet Parasitol 93: 227239.

  • 6.

    Pozio E, Khamboonruang C, 1989. Trichinellosis in Thailand: epidemiology and biochemical identification of the aethiological agent. Trop Med Parasitol 40: 7374.

    • Search Google Scholar
    • Export Citation
  • 7.

    Jongwutiwes S, Chantachum N, Kraivichian P, Siriyasatien P, Putaporntip C, Tamburrini A, La Rosa G, Sreesunpasirikul C, Yingyourd P, Pozio E, 1998. First outbreak of human trichinellosis caused by Trichinella pseudospiralis. Clin Infect Dis 26: 111115.

    • Search Google Scholar
    • Export Citation
  • 8.

    Khumjui C, Choomkasien P, Dekumyoy P, Kusolsuk T, Kongkaew W, Chalamaat M, Jones JL, 2008. Outbreak of trichinellosis caused by Trichinella papuae, Thailand, 2006. Emerg Infect Dis 14: 19131915.

    • Search Google Scholar
    • Export Citation
  • 9.

    Kusolsuk T, Kamonrattanakun S, Wesanonthawech A, Dekumyoy P, Thaenkham U, Yoonuan T, Nuamtanong S, Sa-Nguankiat S, Pubampen S, Maipanich W, Panitchakit J, Marucci G, Pozio E, Waikagul J, 2010. The second outbreak of trichinellosis caused by Trichinella papuae in Thailand. Trans R Soc Trop Med Hyg 104: 433437.

    • Search Google Scholar
    • Export Citation
  • 10.

    Chotmongkol V, Intapan PM, Koonmee S, Kularbkaew C, Aungaree T, 2005. Case report: acquired progressive muscular hypertrophy and trichinosis. Am J Trop Med Hyg 72: 649650.

    • Search Google Scholar
    • Export Citation
  • 11.

    Justus DE, Morakote N, 1981. Mast cell degranulation associated with sequestration and removal of Trichinella spiralis antigens. Int Arch Allergy Appl Immunol 64: 371384.

    • Search Google Scholar
    • Export Citation
  • 12.

    Corpet F, 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Res 16: 1088110890.

  • 13.

    Wu Z, Snabel V, Pozio E, Hurnikova Z, Nareaho A, Nagano I, Takahashi Y, 2007. Genetic relationships among Trichinella pseudospiralis isolates from Australian, Nearctic, and Palearctic regions. Parasitol Res 101: 15671573.

    • Search Google Scholar
    • Export Citation
  • 14.

    Zarlenga DS, Rosenthal BM, La Rosa G, Pozio E, Hoberg EP, 2006. Post-Miocene expansion, colonization, and host switching drove speciation among extant nematodes of the archaic genus Trichinella. Proc Natl Acad Sci USA 103: 73547359.

    • Search Google Scholar
    • Export Citation
  • 15.

    Pozio E, Hoberg E, La Rosa G, Zarlenga DS, 2009. Molecular taxonomy, phylogeny and biogeography of nematodes belonging to the Trichinella genus. Infect Genet Evol 9: 606616.

    • Search Google Scholar
    • Export Citation
  • 16.

    Pozio E, Owen IL, La Rosa G, Sacchi L, Rossi P, Corona S, 1999. Trichinella papuae n.sp. (Nematoda), a new non-encapsulated species from domestic and sylvatic swine of Papua New Guinea. Int J Parasitol 29: 18251839.

    • Search Google Scholar
    • Export Citation
  • 17.

    Owen IL, Sims LD, Wigglesworth MC, Puana I, 2000. Trichinellosis in Papua New Guinea. Aust Vet J 78: 698701.

  • 18.

    Pozio E, Owen IL, Marucci G, La Rosa G, 2005. Inappropriate feeding practice favors the transmission of Trichinella papuae from wild pigs to saltwater crocodiles in Papua New Guinea. Vet Parasitol 127: 245251.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Molecular Identification of Trichinella papuae from a Thai Patient with Imported Trichinellosis

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  • Departments of Parasitology and Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand; Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand; Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Rangsit Campus, Pathum Thani, Thailand; Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

Previously, we reported the presence of imported trichinellosis in a Thai worker returning from Malaysia, who presented with progressive generalized muscle hypertrophy and weakness after eating wild boar meat. This work analyzed a partial small subunit of a mitochondrial ribosomal RNA gene of Trichinella larvae isolated from the patient. The results showed complete identity with a mitochondrial RNA gene of Trichinella papuae (GenBank accession no. EF517130). This is the first report of imported trichinellosis in Thailand caused by T. papuae. It is possible that T. papuae is widely distributed in the wildlife of Southeast Asia.

Human trichinellosis is an important food-borne parasitic zoonosis that causes ~10,000 human infections per year with a corresponding mortality rate of 0.2%.1,2 Humans are infected by eating raw or undercooked meat of various omnivores and carnivores, particularly pork from domestic or wild pigs. In Thailand, the first trichinellosis outbreak was caused by the consumption of raw pork from hill tribe pigs in Mae Hong Son province, in the northern part of Thailand.3 Subsequently, as many as 118 outbreaks were reported from 1962 to 1991, mostly in northern Thailand.4 The sources of infection were mainly hill tribe pigs and wild boars, and the infecting parasite in all cases was regarded as Trichinella spiralis.5 However, species identification confirming the presence of T. spiralis was revealed in only one outbreak.6 Two more species were later found. In 1994, an outbreak of human trichinellosis caused by Trichinella pseudospiralis was reported in Chumporn province in the southern part of Thailand.7 In 2006 and 2007, two outbreaks of trichinellosis caused by T. papuae were reported in Uthai Thani province in the central part of Thailand.8,9 All cases were indigenous in nature.

The first imported case of trichinellosis in Thailand was reported in 2005. A Thai worker with progressive generalized muscle hypertrophy and weakness visited Srinagarind Hospital, Khon Kaen province, in the northeastern part of Thailand10; however, the species was misdiagnosed and not initially identified. The patient had worked in Malaysia and had a history of eating raw hunt wild boar meat 1 month before onset of illness. Quadriceps muscle biopsy was performed by a compression method, and non-encapsulated Trichinella larvae were seen under the microscope (Figure 1). These larvae were isolated and maintained in Swiss mice under hygienic conditions at the Department of Parasitology, Faculty of Medicine, Khon Kaen University, for species identification. The mice were fed with a standard granulated feed and water ad libitum. Handling and processing of animals were performed following the guidelines for animal experimentation of the National Research Council of Thailand. The study was approved by both the Animal Ethics Committee of Khon Kaen University (reference no. 0514.1.12.2/1) and the Human Ethics Committee of Khon Kaen University (reference no. 470655). Informed consent was obtained from the patient. In this study, the worms originally isolated from the patient were subjected to molecular identification, and were found to be Trichinella papuae by polymerase chain reaction (PCR) and sequencing.

Figure 1.
Figure 1.

Muscle biopsy tissue, compressed between glass slides, showing non-encapsulated Trichinella larvae.

Citation: The American Society of Tropical Medicine and Hygiene 84, 6; 10.4269/ajtmh.2011.10-0675

For recovery of T. papuae larvae, the infected mice were killed 2 months after oral inoculation. The infective larvae were regained from mouse muscles by pepsin-HCl digestion, and harvested using a modified Baermann technique.11 Trichinella larvae were washed extensively with normal saline solution. For genomic DNA extraction, the larvae were homogenized with disposable polypropylene pestles (Bellco Glass Inc., Vineland, NJ), and then DNA was extracted using a NucleoSpin Tissue Kit (Macherey-Nagel GmbH & Co., Düren, Germany). The DNA was eluted in 100 μL of distilled water and stored at −20°C until used.

A fragment of a small subunit of mitochondrial ribosomal RNA gene was amplified using the genus-specific primers TPa-F (5′-AATAGTGTGCCAGCTATCG-3′) and TPa-R (5′-CTTCCAGAAGATCTACTTTGTTACG-3′), which were designed from a highly conserved region within a mitochondrial RNA gene of Trichinella (GenBank accession no. EF517130). The PCR was carried out using a GeneAmp PCR System 9700 (Applied Biosystems, Singapore). The reaction was carried out in a 25 μL volume containing PCR buffer (10 mmol/L Tris-HCl, pH 8.4, 50 mmol/L KCl, 1.5 mmol/L MgCl2), 200 μmol/L of each deoxyribonucleotide triphosphate, 0.2 μmol/L of each primer, and 0.625 unit of Taq DNA polymerase (Invitrogen, Carlsbad, CA). The DNA template was initially denatured at 94°C for 5 min. The amplification procedure consisted of 30 cycles at 95°C for 30 sec (denaturation), 55°C for 30 sec (annealing), and 72°C for 30 sec (extension), with a final extension at 72°C for 10 min. Amplified product was run on a 1.5% agarose gel; a 565 bps fragment was then cut and purified for DNA sequencing, which was performed using the MegaBACE 1000 DNA Analysis System (GE Healthcare, Piscataway, NJ). Sequence alignment was conducted using the multiple sequence alignment program of the National Institute for Agricultural Research (INRA) on the MultAlin homepage.12 The partial small subunit of a ribosomal RNA gene of Trichinella larvae Malaysian isolate showed complete identity with a mitochondrial RNA gene of T. papuae (GenBank accession no. EF517130) (Figure 2).

Figure 2.
Figure 2.

Multiple alignment of representative DNA sequence obtained from imported Trichinella Malaysian isolate (Trichinella_pa-ma). Nucleotide sequences from mitochondrial RNA genes of 11 Trichinella species and genotypes—Trichinella papuae, Trichinella zimbabwensis, Trichinella pseudospiralis, Trichinella spiralis, Trichinella nelsoni, Trichinella nativa, Trichinella sp. T6, Trichinella murrelli, Trichinella sp. T9, Trichinella britovi, and Trichinella sp. T8 (GenBank accession nos.: EF517130, EF517131, EF517124, GU339146, EF517127, DQ159093, EF517126, EF517125, EF517129, DQ159092, and EF517128, respectively)—were used for comparison. Dots represent sequence identity with Trichinella Malaysian isolate. Gaps are represented by dashed lines.

Citation: The American Society of Tropical Medicine and Hygiene 84, 6; 10.4269/ajtmh.2011.10-0675

For examination of intraspecies differences existence between geographical isolates, the partial cytochrome oxidase subunit I (COI) region was amplified by specific primers Tri-COIF (forward: 5′-GTTTATATCCTAGTACTACC-3′) and Tri-COIR (reverse: 5′-GCGTTTGATAGTCTAACTCC-3′),8 which were designed from partial COI sequences of mitochondrial DNA from T. papuae (GenBank accession no. DQ007899). The PCR reaction was done similar to the above except the annealing condition at 50°C for 30 sec was performed. After sequencing, the partial COI sequence T. papuae Malaysian isolate revealed complete identity with T. papuae Papua New Guinea isolate (accession no. EF601546)13 and isolate from Thailand (accession no. GQ180179),9 but single nucleotide different with another isolate from Papua New Guinea (accession no. DQ007899).14 This result revealed no intraspecies difference with Thailand isolate (Figure 3).

Figure 3.
Figure 3.

Multiple alignment of partial DNA sequence of cytochrome c oxidase subunit I (COI) gene obtained from four isolates of Trichinella papuae. An imported Trichinella Malaysian isolate (Trichinella_pa-ma) was aligned with three isolates of T. papuae in GenBank (accession nos.: EF601546, DQ007899, and GQ180179). Dots represent the conservation of the base among all the examined isolates. Horizontal bars represent the position of polymerase chain reaction (PCR) primers.

Citation: The American Society of Tropical Medicine and Hygiene 84, 6; 10.4269/ajtmh.2011.10-0675

One encapsulated (T. spiralis) and two non-encapsulated (T. pseudospiralis and T. papuae) species have been identified in Southeast Asia.15 Trichinella papuae was first described in 1999 from domestic and sylvatic swine and farmed saltwater crocodiles of Papua New Guinea.1618 Outbreaks of human trichinellosis caused by T. papuae, however, were subsequently reported in Thailand.8,9 Here, we validated the molecular evidence from our previous study of T. papuae isolated from a Thai worker returning from Malaysia.10 This was the first imported trichinellosis case in Thailand caused by T. papuae. It is possible that T. papuae is distributed in the wildlife of Southeast Asia. People in Singapore who had visited neighboring Malaysian islands in 1998 acquired trichinellosis.1

Prolonged progressive generalized muscle hypertrophy and weakness in a patient, together with a history of consumption of raw wildlife meat, should alert physicians to the possibility of trichinellosis caused by T. papuae.

ACKNOWLEDGMENTS:

We thank Penchome Junwan and Tongjit Thanchomanang for technical support, and are grateful to Yukifumi Nawa for comments and suggestions.

  • 1.

    Dupouy-Camet J, Murell KD, 2007. FAO/WHO/OIE Guidelines for the Surveillance, Management, Prevention and Control of Trichinellosis. Paris: FAO/WHO/OIE, 1119.

    • Search Google Scholar
    • Export Citation
  • 2.

    Gottstein B, Pozio E, Nockler K, 2009. Epidemiology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol Rev 22: 127145.

  • 3.

    Boonthahom P, Nawarat A, 1963. The outbreak of trichinosis at Amphur Mae Sarieng. Bull Public Health 33: 301308.

  • 4.

    Khamboonruang C, 1991. The present status of trichinellosis in Thailand. Southeast Asian J Trop Med Public Health 22 (Suppl): 312325.

  • 5.

    Takahashi Y, Mingyuan L, Waikagul J, 2000. Epidemiology of trichinellosis in Asia and the Pacific Rim. Vet Parasitol 93: 227239.

  • 6.

    Pozio E, Khamboonruang C, 1989. Trichinellosis in Thailand: epidemiology and biochemical identification of the aethiological agent. Trop Med Parasitol 40: 7374.

    • Search Google Scholar
    • Export Citation
  • 7.

    Jongwutiwes S, Chantachum N, Kraivichian P, Siriyasatien P, Putaporntip C, Tamburrini A, La Rosa G, Sreesunpasirikul C, Yingyourd P, Pozio E, 1998. First outbreak of human trichinellosis caused by Trichinella pseudospiralis. Clin Infect Dis 26: 111115.

    • Search Google Scholar
    • Export Citation
  • 8.

    Khumjui C, Choomkasien P, Dekumyoy P, Kusolsuk T, Kongkaew W, Chalamaat M, Jones JL, 2008. Outbreak of trichinellosis caused by Trichinella papuae, Thailand, 2006. Emerg Infect Dis 14: 19131915.

    • Search Google Scholar
    • Export Citation
  • 9.

    Kusolsuk T, Kamonrattanakun S, Wesanonthawech A, Dekumyoy P, Thaenkham U, Yoonuan T, Nuamtanong S, Sa-Nguankiat S, Pubampen S, Maipanich W, Panitchakit J, Marucci G, Pozio E, Waikagul J, 2010. The second outbreak of trichinellosis caused by Trichinella papuae in Thailand. Trans R Soc Trop Med Hyg 104: 433437.

    • Search Google Scholar
    • Export Citation
  • 10.

    Chotmongkol V, Intapan PM, Koonmee S, Kularbkaew C, Aungaree T, 2005. Case report: acquired progressive muscular hypertrophy and trichinosis. Am J Trop Med Hyg 72: 649650.

    • Search Google Scholar
    • Export Citation
  • 11.

    Justus DE, Morakote N, 1981. Mast cell degranulation associated with sequestration and removal of Trichinella spiralis antigens. Int Arch Allergy Appl Immunol 64: 371384.

    • Search Google Scholar
    • Export Citation
  • 12.

    Corpet F, 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Res 16: 1088110890.

  • 13.

    Wu Z, Snabel V, Pozio E, Hurnikova Z, Nareaho A, Nagano I, Takahashi Y, 2007. Genetic relationships among Trichinella pseudospiralis isolates from Australian, Nearctic, and Palearctic regions. Parasitol Res 101: 15671573.

    • Search Google Scholar
    • Export Citation
  • 14.

    Zarlenga DS, Rosenthal BM, La Rosa G, Pozio E, Hoberg EP, 2006. Post-Miocene expansion, colonization, and host switching drove speciation among extant nematodes of the archaic genus Trichinella. Proc Natl Acad Sci USA 103: 73547359.

    • Search Google Scholar
    • Export Citation
  • 15.

    Pozio E, Hoberg E, La Rosa G, Zarlenga DS, 2009. Molecular taxonomy, phylogeny and biogeography of nematodes belonging to the Trichinella genus. Infect Genet Evol 9: 606616.

    • Search Google Scholar
    • Export Citation
  • 16.

    Pozio E, Owen IL, La Rosa G, Sacchi L, Rossi P, Corona S, 1999. Trichinella papuae n.sp. (Nematoda), a new non-encapsulated species from domestic and sylvatic swine of Papua New Guinea. Int J Parasitol 29: 18251839.

    • Search Google Scholar
    • Export Citation
  • 17.

    Owen IL, Sims LD, Wigglesworth MC, Puana I, 2000. Trichinellosis in Papua New Guinea. Aust Vet J 78: 698701.

  • 18.

    Pozio E, Owen IL, Marucci G, La Rosa G, 2005. Inappropriate feeding practice favors the transmission of Trichinella papuae from wild pigs to saltwater crocodiles in Papua New Guinea. Vet Parasitol 127: 245251.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to Wanchai Maleewong, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. E-mail: wanch_ma@kku.ac.th

Financial support: This research was supported by grants from the National Science and Technology Development Agency (Discovery Based Development Grant); the Office of the Higher Education Commission; the National Research Council of Thailand; and Khon Kaen University (National Research University Project), Thailand.

Authors' addresses: Pewpan M. Intapan, Oranuch Sanpool, and Wanchai Maleewong, Department of Parasitology, Faculty of Medicine, and Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand, E-mails: pewpan@kku.ac.th, chimy_6891@hotmail.com, and wanch_ma@kku.ac.th. Verajit Chotmongkol, Department of Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, E-mail: chotmongkolverajit@yahoo.com. Chairat Tantrawatpan, Research and Diagnostic Center for Emerging Infectious Diseases, Khon Kaen University, Khon Kaen, Thailand; and the Division of Cell Biology, Department of Preclinical Sciences, Faculty of Medicine, Thammasat University, Rangsit Campus, Pathum Thani, Thailand, E-mail: talent3003@yahoo.com. Nimit Morakote, Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand, E-mail: nimit@med.cmu.ac.th.

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