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The ﬁrst occurrence of Trichinella murrelli in wild boar in Iran and a review of Iranian trichinellosis
E.B. Kia1*, A.R. Meamar2, F. Zahabiun1 and H. Mirhendi1
Department of Medical Parasitology and Mycology, School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences, Tehran, Iran: 2Department of Medical Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
(Accepted 3 March 2009; First Published Online 17 June 2009) Abstract Trichinella larvae isolated from the thigh muscle of a wild boar, Sus scrofa, captured from Gilan Province, northern Iran, was processed for DNA analysis. Polymerase chain reaction (PCR) for ampliﬁcation of the 5S rDNA fragment demonstrated a 700 bp band on agarose gel. Analysis of DNA sequencing by BLAST conﬁrmed the isolate as T. murrelli. This report constitutes the ﬁrst recorded occurrence of T. murrelli in Asia, and also the ﬁrst occurrence in a wild boar host. Introduction
Trichinellosis is a zoonosis, caused by ingestion of raw or undercooked meat containing larvae of the nematode genus, Trichinella. Species of Trichinella can be found worldwide in a variety of hosts, ranging from mammals to birds and reptiles. Wildlife acts as a natural reservoir for Trichinella. Links between the sylvatic and domestic cycle are well reported and, depending on human cultural food practices, the causative agent may enter the human food chain, resulting in infection (Pozio & Murrell, 2006). The genus Trichinella is divided into an encapsulated clade and a non-encapsulated clade, referring to the collagen capsule that surrounds the larvae of some species in the host muscle. Currently, the genus Trichinella is classiﬁed into eight species (T. spiralis, T. nativa, T. britovi, T. pseudospiralis, T. murrelli, T. nelsoni, T. papuae and T. zimbabwensis) and three unclassiﬁed genotypes (Trichinella T6, Trichinella T8 and Trichinella T9) (Pozio & Zarlenga, 2005; Rodriguez et al., 2008). All known Trichinella species are morphologically indistinguishable except for T. pseudospiralis, which is smaller in size than other species. The geographical distribution of the different species is related to ecology, climate and host behaviour (Pozio et al., 1992; Kapel, 1997; Muller, 2002).
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The identiﬁcation of sequence regions in the genomes of pathogens, which can be useful to distinguish among species and genotypes, is of great importance for epidemiological, molecular and phylogenetic studies. The 5S ribosomal DNA intergenic spacer region has been identiﬁed as a good target to distinguish the eight Trichinella species and genotypes (Rombout et al., 2001; De Bruyne et al., 2005; van der Giessen et al., 2005). This method is simple (only one primer pair), sensitive (DNA from a single larva sufﬁces) and inexpensive (low-cost sequencing is available). It can also be applied directly to muscle biopsy specimens (Rombout et al., 2001). In Iran, Trichinella has been reported in different wild reservoir hosts. In the northern part of Iran, it has been reported in wild boars (Sus scrofa) (Afshar & Jahfarzadeh, 1967; Mobedi et al., 1973), jungle cats (Felis chaus) (Mobedi et al., 1973), brown bears (Ursus arctos) (Mobedi et al., 1973), golden jackals (Canis aureus) (Mobedi et al., 1973; Hamidi, 1977; Massoud & Mahdavi, 1987; Dalimi & Mobedi, 1992), red foxes (Vulpes vulpes) (Hamidi, 1977) and stray dogs (Hamidi, 1977); in Ardabil Province, north-western Iran, it has been identiﬁed in golden jackals and red foxes (Zariffard, 1995); in Isfahan, central Iran, in golden jackals, red foxes, stray dogs, hyena (Hyaena hyaena) and one species of rodent (Meriones persicus) (Sadighian et al., 1973); in Khuzestan Province, southwestern Iran, it has been found in mongoose (Herpestes auropunctatus) (Mowlavi et al., 2000); in Bandar Abbas,
. molecular weight marker. red foxes. The ﬁrst of these was a single case in 1966.48 mm and 36. There has also been a second. Parasitological examinations Part of the muscle tissue was prepared for direct microscopic examination and Trichinella encapsulated larvae were observed. This also constitutes the ﬁrst recorded occurrence of this species in Asia. followed by a 5 min extension step at 728C. The amplicon was electrophoresed on 1. In spite of the occurrence of infection in various animal hosts from different parts of the country. negative control. northern Iran in April 2007. In addition. with a worm burden of 7 larvae/g.400 E. stray dogs (Hamidi & Mobedi. human infectivity has been documented twice. The PCR reaction. using speciﬁc primers. Gilan Province. DNA extraction The parasite DNA was extracted using QIAamp DNA Mini kit (Qiagen. Electrophoresis of PCR products of Trichinella murrelli larvae isolated from wild boar. in both cases through consuming wild boar meat. a piece of muscle was preserved in 10% formalin and. The patients were six household members living in Tehran who consumed semi-cooked meat of the described muscle as kebab. Sequencing The PCR product was sequenced by SEQLAB company (Germany) with an ABI 3100 genetic analyser. 1973. Siahkal. The nucleotide sequence was aligned with those from the GenBank database. 1).5 mM MgCl2. south of Iran. more recent single-source outbreak (Kia et al.5 U of Taq polymerase (Roche. 1973.B. 1977. Reactions were performed in a 50 ml Discussion Trichinella circulates in a sylvatic cycle in various wild animals (Afshar & Jahfarzadeh. 948C for 30 s. history of the disease and serology (Moin. murrelli was submitted to the Genebank/EMBL/DDBJ database under Accession Number AB426627. In the histological sections of the muscle. and 3. Hamidi & Mobedi. Mobedi et al. the size (length and width) of ten larvae was measured. The average length and width of ten larvae after pepsin – HCl digestion of the muscle was 1083. using BLAST software. Twenty gram of thigh muscle was digested by a pepsin– HCl method. 1966). 5SF (50 -GGA-ACA-CGC-AGT-GTCGTA-GAC-30 ) and 5SR (50 -TGG-TAT-GAT-CGT-AGACAT-TTG-C-30 ). No infection has so far been reported in domestic animals. After separation of larvae from the digestion ﬂuid by washing three times in distilled water. in golden jackals. 1.4 mm respectively. Mannheim. Materials and methods Source of sample The source of the sample was part of a frozen thigh muscle of a wild boar hunted in Javaher Dasht forest. Lanes: 1. . yielded a 700 bp band on agarose gel (ﬁg.5 mM of each primer and 5 ml DNA sample. Hamidi. the sequence was analysed by BLAST and the isolate was identiﬁed as T. surrounded by an inﬂammatory reaction. personal communication). 2. 1977) and recently in a leopard (Panthera pardus) (Mowlavi. 1. Polymerase chain reaction A 700 bp region of the 5S ribosomal DNA gene was ampliﬁed by polymerase chain reaction (PCR) with two designed primers.. After gel extraction of the PCR product and DNA sequencing. Germany). The muscle was minced by scissors and incubated in artiﬁcial gastric juice for 24 h at 378C. Germany) according to the manufacturer’s instructions for tissue extraction. The nucleotide sequence of T. encapsulated Trichinella larvae were observed. murrelli. 200 mM of each dNTP. 1967.. Hilden. 5 mm sections were prepared and stained with haematoxylin and eosin. This study presents a molecular description of Trichinella murrelli isolated from a wild boar in northern Iran. no molecular description of the species has been presented prior to this work. which constituted the ﬁrst report of trichinellosis in the country and was diagnosed based on clinical symptoms. 0. Fig. Results Trichinella larvae were visible inside thick collagen capsules by direct microscopic examination of muscle tissue. Tehran University of Medical Sciences in May 2007 by patients who had characteristic symptoms of trichinellosis and were referred for the diagnosis of their infection. Kia et al.5% agarose gel and visualized under an ultraviolet transilluminator after staining with ethidium bromide. Trichinella murrelli. The sample was delivered to the helminthological laboratory of the School of Public Health. 578C for 90 s and 728C for 45 s for 35 cycles. Sadighian et al. reaction containing 1. Reactions were run in a Perkin-Elmer Thermocycler under the following conditions: 958C for 6 min for one cycle. 2008). following tissue processing by conventional histological methods. However.
M. USA (Pozio. with only one nucleotide difference compared to T. murrelli (Ancelle et al. Boireau. P. (1992) Helminth parasites of carnivores in northern Iran.. Acknowledgements The authors would like to acknowledge the help received from Mrs M. Zariffard. J. D. P. J. J. Tehran University of Medical Sciences and Dr E. Later.P. murrelli.P. 57 – 61. Hamidi. & Mobedi. using DNA-based methods for identiﬁcation of the species involved is necessary. based on the 5S rDNA sequences of the best-known Trichinella species. to the best of our knowledge.. 2000) were reported as T. isolates from golden jackal (Massoud & Mahdavi.. H.E. H. 2008). revealed symptoms characteristic of trichinellosis.T. Illinois. D. respectively (Kia et al. Dupouy-Camet. murrelli from Nearctic America (Accession No.. Sharbatkhori. De Bruyne. prior to this work. Mowlavi et al. References Afshar. Despite this. including fever. New Mexico.. Hill. caused by T. Georgia. E. However. previously determined by Rombout et al. Massoud & Mahdavi. 1988). the average eosinophil count was 2091 mm3. 1992.. Trichinella murrelli was considered as a predominant aetiological agent of sylvatic trichinellosis exclusively in temperate regions of North America (Pozio. 2008). Nozais. This study is the ﬁrst research on the molecular characterization of Trichinella in Iran. J. A...E. A. murrelli in Asia and. 2001)..S. Le Guerhier. Dubey. nelsoni. 1977. & Dupouy-Camet. the isolate of Trichinella detected from wild boar in the temperate area of northern Iran was identiﬁed as T. spiralis. bobcats and in one horse. raccoons. Since Trichinella has been detected in various wild animal hosts from geographic areas with different climates in the country.... Dupouy-Camet... J. Annals of Tropical Medicine and Parasitology 61. 2000) in different parts of Iran. (2005) Simple species identiﬁcation of Trichinella isolates by ampliﬁcation and sequencing of the 5S ribosomal DNA intergenic spacer region. Apart from the abovementioned studies. all ranging from 24 to 27 years old.. (1977) Trichiniasis among the animals in North Eastern Iran (1). murrelli in wild boar in Iran 401 1977. 349– 351. AY009947). 14 – 19. Virginia. in Connecticut. in Khuzestan Province. murrelli from around the world and comparison of its clinical patterns and pathogenicity in humans with the other Trichinella species will provide a better understanding of this zoonotic disease and improve its diagnosis and treatment. also the ﬁrst report of occurrence of T. Nevertheless. 1302–1311. F. and the average creatine phosphokinase and lactic dehydrogenase enzyme values were 890 U/l (N ¼ 225– 500) and 584 U/l (N ¼ 25– 200). It was the cause of moderate trichinellosis in humans. (2001) Trichinella murrelli: pathological features in human muscles at different delays after infection. (1967) Trichinosis in Iran. (2008) An outbreak of human Trichinellosis due to consumption of boar meat . simultaneously. S176–S179.N. A. Parasite 8. Z. (1988) Two outbreaks of trichinosis caused by horsemeat in France in 1985. murrelli in wild boar in the world. 1969. V. This is the ﬁrst report of T. G. 395– 397. Integration of the ﬁndings from different human cases of T. 1987. Dalimi & Mobedi.. Veterinary Parasitology 132. T. & Kordbacheh. Hamidi. Bulletin de la ´te ´ de Pathologie Exotique et de ses Filiales 72. Ancelle. A. sequencing and comparison with the published data. 1966. Zahabiun. In conclusion. Razmjou. Shaikenov & Boev (1983) cross-bred four Trichinella isolates from golden jackal from the north and south-west of Iran with standard strains. A. & Jahfarzadeh. Although. Lavarde. & Mobedi. 2006). 1987) and mongoose (Mowlavi et al. I. Iran University of Medical Sciences. Veterinary Parasitology 137. ethnic minorities and hunters who practise consumption of raw or undercooked meat of wild pork are at risk of trichinellosis. Kia.. nelsoni and T. (2001).. Dalimi. (1977) Sylvatic focus of Trichiniasis in Bandar abbas area South of Iran.. even with a low larvae burden in consumed meat. Meamar.. T. 1995. no molecular description of the species involved in wild animal or human infectivity has been presented. Soodbaksh. In three of the patients. American Journal of Epidemiology 127. 2001). Yera. no other studies reporting trichinellosis in wildlife have attempted to characterize Trichinella species. Petit.R. black bears. 1976. USA (Dupouy-Camet et al. Iranian Journal of Public Health 6. the Scandinavian countries and the Baltic States. & Zarlenga. 30 – 33. Indiana. G. USA (Dubey et al. The method used combined PCR-based ampliﬁcation.B. 374– 378. Faculty of Medicine. Kapel. It has been detected in red foxes. in two separate studies based on sensitivity of laboratory animals (white mouse and rat) to Trichinella. murrelli is circulating in a sylvatic cycle in the temperate region of northern Iran. it was later concluded that the origin of the horse carcass was from Connecticut. F. This is mainly due to Muslim beliefs based on not consuming pork and the meat of some other animal species. and recently in a domestic dog in Chase City.. A. Among the seven people who had consumed semicooked meat of the relevant boar. I. School of Public Health. & Lapierre. C. De Pinieux. A. nelsoni and those from the south as T. J.. The newly studied T. for whom blood samples could be taken. coyotes. making it important for public health. six people. subarctic and temperate regions: Greenland. (2006) A Trichinella murrelli infection in a domestic dog in the United States. Southeast Asian Journal of Tropical Medicine and Public Health 28. V. 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