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The occurrence of Contracaecum sp. larvae (Nematoda: Anisakidae) in four teleostean species from Lake Nasser, Egypt: morphological and molecular studies
The Journal of Basic and Applied Zoology volume 78, Article number: 9 (2017)
Nematodes of the family Anisakidae are parasites of many fishes and aquatic invertebrates which act as intermediate or paratenic hosts, while mammals and fish-eating birds are definitive hosts. Infective L3 larvae may be incidentally taken by human through eating raw or undercooked fish meat, causing anisakidosis. The main purpose of this study is to provide a basis for the future investigations to discover the genetic diversity of this widely distributed parasite nematodes of fishes and fish eating animals and their effect on fisheries and public health in Egypt and worldwide.
One thousand, one hundred and fifteen specimens belong to nine fish species were collected from Lake Nasser, Egypt, and examined for infection with Anisakid larvae. Four fish species (Oreochromis niloticus, Tilapia galilaea, Lates niloticus, and Hydrocynus forskahlii) were found infected with third stage larvae of Contracaecum spp. No other Anisakid nematodes were detected. Larvae were found in the body cavity adhering to mesenteries by a thin membrane, except in Oreochromis niloticus and Tilapia galilaea were found free in branchial chambers. The highest prevalence was recorded in L. niloticus (100%) and H. forskahlii (82%). The mean intensity of infections were 0.17–4.12 and 5.1–10.3 in L. niloticus and H. forskahlii respectively. For further identification, the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA from isolated larvae (n = 54) were amplified by PCR, followed by single-strand conformation polymorphism (SSCP) analysis which revealed five possible profiles.
Light and scanning electron microscope studies revealed that all anisakid larvae in the present study showed the most typical features of the genus Contracaecum. The sequencing (n = 28) and sequence and phylogenetic analyses showed that the present nematode larvae are likely belonging to C. multipapillatum.
Parasites usually influence the quality and marketing of commercially produced fish and may contribute to high fish mortalities and economic losses or threaten the abundance and diversity of fish species, accordingly, raising a lot of public health concerns, particularly in regions where raw or smoked fish are eaten (Adams, Murrell, & Cross, 1997; Paperna, 1996; Barson, 2004).
Nematodes of the family Anisakidae are parasites of many fishes and aquatic invertebrates which acting as intermediate or paratenic hosts, while mammals and fish-eating birds are definitive hosts. The most widespread genera are Anisakis, Pseudoterranova, and Contracaecum which have similar life cycles. Third stage larvae (L3) of Contracaecum (Railliet et Henry, 1912) were found usually in the body cavity, branchial chambers, and mesenteries of the fish while the adults were found in the gut of the fish-eating birds as mentioned by Whitfield and Heeg (1977). It was reported in Cichlids and Catfish in different African countries, such as Egypt (Amin, 1978), East Africa (Aloo, 2001) and South Africa (Boomker, 1982, 1994; Van As & Basson, 1984). These larvae were the most prevalent nematode parasite identified from the mesenteries of Clarias gariepinus, Barbus acutirostris, B. tsanensis, and B. brevicephalus and from the pericardial cavity of Nile Tilapia (Oreochromis niloticus) captured from Lake Tana Ethiopia (Yimer & Enyew, 2003). Kaddumukasa, Kaddu, and Makanga (2006) investigated the positive correlation between intensity of infection and length of Nile tilapia, O. niloticus, in Lake Wamala. The high spread of Contracaecum in fish might influence their health; subsequently, it can affect the commercial value of fish, particularly when it was found in the musculature, and thus represent some economic loss for fisheries industry (Angot & Brasseur, 1995). At the point when in the stomach of the definitive host, L3 molts to become L4 and both the larvae and the adults may influence the host health negatively (Martins, Onaka, & Fenerick Jr., 2005; Girişgin, Alasonyalilar-Demirer, & Girişgin, 2012). In case of the larvae are incidentally taken by human through eating raw or undercooked fish meat, they may cause anisakidosis, a zoonotic infection causing stomach pains, fever, diarrhea, and vomiting (Sakanari & McKerrow, 1989; Kaneko, 1991; Arslan, Dinçoğlu, & Güven, 1995; Audicana, Ansotegui, de Corres, & Kennedy, 2002; Palm, 2004; Shamsi & Butcher, 2011).
The identification of Contracaecum spp. from various host groups attracted attention of scientists in different geographical parts of the world using both light and scanning electron microscopy for morphological examination, as well as the evaluation of genetic markers as the ribosomal internal transcribed spacers (ITS-1 and ITS-2) for the molecular characterization (Nadler, D'Amelio, Dailey, Paggi, Siu, et al., 2005; D'Amelio, Barros, Ingrosso, Fauquier, Russo, et al., 2007; Shamsi, Gasser, Beveridge, & Shabani, 2008; Shamsi, Norman, Gasser, & Beveridge, 2009; Shamsi, Gasser, & Beveridge, 2011; Garbin, Mattiucci, Paoletti, González-Acuña, & Nascetti, 2011; Garbin, Mattiucci, Paoletti, Diaz, Nascetti, et al., 2013; Jabbar, Fong, Kok, Lopata, Gasser, et al., 2013; Borges, Santos, Brandã, Santos, Miranda, et al., 2014).
The Fishes are an important source of protein for humans and other animals. The fish industry likewise provides livelihood chances to numerous individuals, as well as income at household and national levels (FAO, 1996).
Lake Nasser is a reservoir constructed by the High Dam in southern part in Egypt and extends to the second cataract in Sudan. It is about 480 km long, 300 km in Egypt (Lake Nasser), and 180 km in Sudan (Lake Nubia). Lake Nasser is considered as important source of fish and of considerable economic potential in Egypt. Its estimated commercial value of the fishery (including both fresh and salted fish) is around US $17 million (Béné, Bandi, & Durville, 2008).
Fifty-seven species of fish belonging to 16 families were recorded in Lake Nasser. Tilapia nilotica (Oreochromis niloticus), Nile perch (Lates niloticus), Tiger fish (Hydrocynus forskahlii), and Alestes spp. are the most common species and important for the fishery (Latif, 1974; Rashid, 1995). While Tilapia and Nile perch are landed fresh, Tiger fish and Alestes are usually processed before being sold as salted fish (Bishai, Abdel-Malek, & Khalil, 2000).
However, the parasitic fauna of fishes from different inland water in Egypt were studied by several investigators; only few published data based on morphological characteristics were performed on helminth infections in Lake Nasser (Saoud & Wannas, 1984; Al-Bassel, 1992; Saad, 2007; Ezzat, ElKorashey, & Sherif, 2012). Thus, our knowledge about the prevalence, abundance, and the genetic diversity of nematodes of fishes is still limited. The present investigation is the first study to use an integrated molecular and morphological approach toward identification and detailed description of socioeconomically important anisakid nematodes from four infected out of nine examined species of fishes in Lake Nasser, Egypt.
Guidelines and rules of Aswan University were followed in terms of dealing with and use of animals. Approvals from the committee of Graduate Studies and from the College Council were taken before starting the study.
During 2 years, from November 2013 to October 2015,1115 fish specimens were collected from Lake Nasser, Egypt, and examined for infection with anisakid larvae. These fishes were Oreochromis niloticus (n = 73); Tilapia galilaea (n = 712); Lates niloticus (n = 85); Synodontis frontosus (n = 40); Mormyrus caschive (n = 53); Chrysichthys auratus (n = 43); Hydrocynus forskahlii (n = 35); Alestes baremose (n = 44); and Lebeo niloticus (n = 30). They were caught from 6 different localities (Khors) of the Lake, Allaqi, Dehmet, Khor Ghazal, Khor Maria, Khor Galal, and Wady abyad. Then, they were immediately transported to the laboratory for parasite examinations. Subsequently, they were dissected and the alimentary tract, liver, and gills were taken out and examined in physiological saline. Also, the body cavity was examined for investigation of nematode infection. The worms were collected and washed extensively with physiological saline. The prevalence and intensity were calculated for each fish species.
Each individual nematode was divided into three pieces. Small pieces of the middle parts were kept in − 20 in 70% ethanol for molecular studies, while the anterior and posterior parts fixed in hot 70% ethanol (60–70 °C) for relaxing their bodies, and then preserved in 70% ethanol with few drops of glycerin to avoid distortion of the outer layer of the cuticle. Before light microscopic study, worms were cleared in Lactophenol for 24 h. All measurements were made specifically with an eyepiece micrometer and given in millimeters, unless mentioned otherwise. The photomicrographs were taken for the obtained nematodes using Olympus light microscope attached with a camera model HBS (HB2) and digital microscope model Olympus CX41. Identifications were based on key features and descriptions (Yamaguti, 1961; Nadler & Hudspeth, 1998; Anderson, 2000; Martins et al., 2005; Olivero-Verbel, Baldiris-Avila, Guette-Fernandez, Benavides-Alvarez, Mercado-Camargo, et al., 2006). For the scanning electron microscope (SEM) examinations, specimens were fixed in 5% glutaraldehyde solution for 24 h and then washed five times in Sodium cacodylate buffer solution (15 min each). Post-fixation, specimens were rinsed in 1% of osmic acid for 2 h. After washing, three changes in Sodium cacodylate buffer solution (15 min each) took place, and then the specimens were dehydrated in ascending grade of alcohols; 30, 50, 70, 90, and 100% alcohol for 30 min each. Then, the specimens were dried to acritical point as a result of exposure to 25 °C. Subsequently, samples were placed on holder and were coated by a very thin layer of gold (thickness of 150–200 Angstroms). Finally, the specimens were examined by scanning the electron microscope type JSM 5400 LV: Jeol.
Genomic DNA isolation, PCR, and sequencing
Genomic DNA was isolated from individual larvae (n = 54), as described before (Younis, Geisinger, Ajonina-Ekoti, Soblik, Steen, et al., 2011) with slight modifications. In brief, the individual parasite materials were digested for 3–4 h at 56 °C with proteinase K in ALT buffer (Dneasy kit, Qiagen) under a constant agitation. gDNA extracted with the standard phenol⁄chloroform method and treated with RNase A was then precipitated with 5.2 M ammonium acetate and dissolved in 20–30 μl HPLC water. Concentrations were determined by spectrophotometry.
The specific primer sets SS1/NC13R and SS2/NC2 were used to amplify the two nuclear ribosomal markers ITS-1 and ITS-2 respectively, under the same conditions as described previously (Zhang, Hu, Shamsi, Beveridge, Li, et al., 2007). The negative control (gDNA from intact fish muscles and non-DNA) samples were used. The PCR products were analyzed by 1% w/v agarose gel, stained with ethidium bromide and photographed using a gel documentation system, were then purified using QIAquick® PCR purification kit (Qiagen), according to the manufacturer’s protocols. The resulted products were sequenced by a dideoxy termination method using an applied Biosystems 373 DNA sequencer (LGC Genomics GmbH, Germany) in the two directions (forward and reverse) by the same PCR-used primers.
Single-strand conformation polymorphism (SSCP)
All amplicons (ITS-1 and ITS-2) were subjected to SSCP analysis (Gasser, Hu, Chilton, Campbell, Jex, et al., 2006) to screen nucleotide sequence variation among amplicons. In details, 5 μl of individual amplicons were mixed with 10 μl of loading buffer (75% formamide, 2.5 mM Tris, 0.0075 bromophenol blue, 0.0075% xylene cyanol, 15% glycerol, 15 mM EDTA), denaturated at 96 °C for 15 min and snapped cooling on a freeze block (− 20 °C) for 5 min. Individual samples (15 μl) were loaded into the wells of a 10% acrylamide/bis gel (29:1), containing 7% glycerol, and 1X TBE buffer and casted in mini protein tetra vertical chamber (Bio-Rad). Gels were subjected to electrophoresis for 7 h at constant power (70 V) and temperature 8 °C in a pre-cooled 1XTBE. The gels were then stained with ethidium bromide (1 μg/ml in 1XTBE buffer for 15 min), de-stained in 1XTBE for 10 min and photographed by an ultraviolet transilluminator.
Computer-based sequence analysis
For each sequence, the NCBI Blast program was used for homology search (http://www.ncbi.nlm.nih.gov/). After that, each sequence (forward) was compared to its complement (reverse) and then adjusted manually to get one sequence. Subsequently, the resulted sequences were aligned to each other and to the most homologous sequences in the database using CLUSTALW multiple sequence alignment program. The phylogenetic trees were constructed using online tool software Phylogeny.fr program (Dereeper, Guignon, Blanc, Audic, Buffet, et al., 2008).
Morphological examinations and measurements showed that all the nematodes larvae examined in the present study showed the most typical features of L3 of the genus Contracaecum with convergent shapes and morphology. Interestingly, there were no significant morphological differences of larvae among the fish species, with exception that there were two forms of L3 infected L. niloticus and H. forskahlii, short form (SF) and long form (LF).
All larvae were found to share most of morphological characters with slight differences (Table 1; Figs. 1, 2, and 3). Living larvae were reddish-yellow in color. The body was long, cylindrical with rounded anterior end and tapered posterior one. Light and scanning electron microscopes data can be summarized as follows: the mouth is triangular or transverse in shape and surrounded by three lips, one dorsal and two ventrolateral; lips are provided with four papillae, two on the dorsal lip and one on each ventrolateral lip; well-defined boring tooth is located in between the ventrolateral lips; excretory pore is located anteriorly just below to the boring tooth; length of esophagus, ventricular appendix, intestinal caecum, and the ratios between each other are not the same; anal opening is located near the posterior end; the length of tail is also not equal between larvae collected from different fish species; and tail is blunt in larvae collected from Oreochromis niloticus and T. galilae, while being conical in larvae collected from other fishes and tail ending with a tapered process like spine.
Regarding SEM study, the cuticle is striated with regular and irregular rings, which are narrower anteriorly and become wider as extended posteriorly and showing different ornamentations between larvae where cuticle of collected larvae from Oreochromis niloticus and T. galilae is striated transversely and longitudinally and forming well-defined circular ridges, which are very close anteriorly and covered with heavy mucus derived from the host (Fig. 2a, c). Larvae collected from Lates niloticus present in two sizes: short form (SF) and long form (LF). They are decorated with wavy striations in SF and net shaped in LF as shown in Fig. 4(b, c). Larvae (SF and LF) found in Hydrocynus forskahlii were striated transversely and longitudinally with overlapping strips (dendritic shape) respectively as shown in Fig. 4(d, e).
Prevalence and infection intensity
Nine species of Lake Nasser fishes were examined for infection with Contracaecum larvae. Four fish species (Oreochromis niloticus, Tilapia galilaea, Lates niloticus, and Hydrocynus forskahlii) were found infected with L3 of Contracaecum, and the other five fish species (Synodontis frontosus, Mormyrus caschive, Chrysichthys auratus, Alestes baremose, and Lebeo niloticus) were found free in infection. Contraceacum L3 was found free in pericardial cavity and branchial chambers (O. niloticus and T. galilaea) and in the body cavity adhering to the alimentary canal (L. niloticus and H. forskahlii). The worm burden, prevalence and mean intensities per infected fish varied among different fishes (Table 2), with 100% prevalence in L. niloticus and the highest intensity in H. forskahlii. Interestingly, there were no significant differences in the data collected from different Khors of the Lake Nasser.
PCR of ITS-1 and ITS-2 and mutation scanning analysis
Following the morphological identification, the ITS-1 and ITS-2 regions were amplified by PCR from genomic DNA samples (n = 54) from individual larvae. Agarose gels analyses revealed the same size for each ITS region. Amplicons were ~ 530 bp and ~ 430 bp for the ITS-1 and ITS-2, respectively (Fig. 5a ), confirming that all sequences are of the same genus. Following, all ITS-1 and ITS-2 amplicons were analyzed using SSCP analysis to scan for sequence variability within and among individual specimens. For each ITS region, five possible profiles were observed on the SSCP gels for all 54 samples (Fig. 5b, c). Selected amplicons (n = 28) representing each possible genotype, taking fish species into consideration, were subjected to sequencing. No polymorphism was detected in any sequences that have the same SSCP profile.
Sequence and phylogenetic analyses
Alignment of resulted sequences revealed considerable variation of each ITS region, which may indicate the presence of more than one type of larvae. Scores of identities between ITS-1 sequences were ranging from 65% (between LF larvae collected from L. niloticus and H. forskahlii) to more than 99.3% (between SF larvae collected from L. niloticus and H. forskahlii), while scores of identities between ITS-2 sequences were ranging from 42% (between LF larvae collected from H. forskahlii and larvae from T. galilaea) to more than 98.7% (between larvae collected from O. niloticus and larvae from T. galilaea) (Fig. 6).
The Blast analyses revealed that all sequences of both ITS-1 and ITS-2 in the present study were different from all sequences deposited in GenBank databases. ITS-1 sequences of Contracaecum larvae obtained were deposited in GenBank under accession numbers KX580602-KX580607, and ITS-2 sequences from the corresponding larvae were deposited in GenBank under accession No. KX580607-KX5806013. ITS-1 sequences obtained from larvae-infected T. galilaea showed the highest similarity (89%) to ITS-1 sequence of L3 of Contracaecum sp. 1 (Accession numbers KF990491), isolated from the body cavity and the intestines of H. forskahlii and T. zillii in Lake Turkana, Kenya (Otachi, Szostakowska, Jirsa, & Fellner-Frank, 2015). The last in turn draw an analogy (89%) to the Contracaecum multipapillatum (von Drasche, 1882) from the Australian pelican Pelecanus conspicillatus (Accession numbers AM940056) (Shamsi et al., 2008). In addition, ITS-2 from the same larvae showed the highest similarity (86%) to three ITS-2 sequences of Contracaecum L3 (Accession numbers FM210437- FM210438- FM210439) from Iranian fish (recorded by Shamsi in 2008 to the GenBank). Similarly, sequences of ITS-1 and ITS-2 of L3 from O. niloticus showed 99 and 86% similarity to Contracaecum multipapillatum (Accession numbers KF990491) and to Contracaecum L3 (Accession numbers FM210437-FM210439) respectively. In H. forskahlii, two forms (differ in size) of larvae were found, SF and LF. The last is morphologically similar to all other larvae found in other fishes. ITS-1 of SF larvae collected from H. forskahlii showed the highest similarity (97%) to three ITS-1 sequences (Accession numbers FM210433- FM210434- FM210435) from C. multipapillatum L3 which were collected from intestine and body cavity of barboid fishes in Parishan Lake, Iran (Shamsi & Aghazadeh-Meshgi, 2011). Further, ITS-2 of the same larvae (SF L3 from H. forskahlii) showed the highest similarity (98%) to the previously abovementioned three ITS-2 sequences of Contracaecum L3 (Accession numbers FM210437-FM210438-FM210439). Also, ITS-1 of LF larvae collected from H. forskahlii showed the highest similarity (78%) to the three abovementioned ITS-1 sequences of C. multipapillatum L3 (Accession numbers FM210433- FM210434- FM210435). Differently, ITS-2 sequence of the same larvae (LF L3 from H. forskahlii) showed the highest similarity (92%) to 18S- 5,8S-, 28S-rDNA-cluster of Contracaecum osculatum (Rudolphi, 1802) larvae (Accession numbers KM273050) in cod (Gadus morhua) from the Baltic Sea (Mehrdana, Bahlool, Skov, Marana, Sindberg, et al., 2014). Moreover, L. niloticus had SF and LF larvae. ITS-1 of SF larvae collected from L. niloticus showed the highest similarity (98%) to the three abovementioned ITS-1 sequences of C. multipapillatum L3 (Accession numbers FM210433- FM210434- FM210435). Also, ITS-2 of the same larvae (SF L3 from L. niloticus) showed the highest similarity (98%) to the abovementioned three ITS-2 sequences of Contracaecum L3 (Accession numbers FM210437-FM210438-FM210439). Finally, ITS-1 of LF larvae collected from L. niloticus showed the highest similarity (92%) to the three abovementioned ITS-1 sequences of C. multipapillatum L3 (Accession numbers FM210433- FM210434- FM210435). In addition, ITS-2 of SF L3 from L. niloticus showed the highest similarity to the abovementioned three ITS-2 sequences of Contracaecum L3 (Accession numbers FM210437-FM210438-FM210439).
Phylogenetic analysis for both ITS-1 and ITS-2 sequences produced nearly similar results, of which the first is displayed in Fig. 7. The identities and phylogenetic distances of the selected and most related ITS sequences showed nematode larvae investigated in this study are closely related to C. multipapillatum and likely are of different sub-species.
The present investigation is the first study to use integrated molecular and morphological approaches toward characterization of larval anisakid nematodes from four infected out of nine examined species of fishes in Lake Nasser, Egypt.
Based on morphological characteristics, all anisakid larvae collected from O. niloticus, Tilapia galilaea, Lates niloticus, and Hydrocynus forskahlii fishes in present study are belonging to the genus Contracaecum. They were found in the body cavity adhering to the mesentery by a thin membrane, except in O. niloticus and Tilapia galilaea were found free in pericardial cavity and gills. It was noticed that the parasitic fauna of Lake Nasser is changeable and there was a peak of infection by this larvae in fish species. Al-Bassel (1990) found the juveniles of Contracaecum sp. in the abdominal and branchial cavities of a variety of Lake Nasser fish species, such as Clarias gariepinus, Barbus bynni, Lates niloticus, Synodontis schall, Sarotherodonl galilaeus, and Mormyrus kannume. Garo (1993) reported that such juveniles were found in the branchial cavity and mainly in the sinus venosus of 51.6% of Serotherodon galilaeus fish from Lake Nasser. This fluctuation in the parasitic fauna of Lake Nasser may be a result of the change of water level, temperature degrees of the Lake, and the intensity of migratory birds. It may also be owing to the increasing numbers of intermediate and paratenic hosts and the change of the habitat of these hosts. The reports of Skov, Kania, Olsen, Lauridsen, and Buchmann (2009) and Mathenge (2010) proved that the infection in wild fish was higher than farmed fish due to the high abundance of definitive piscivorous birds in the wild regions; also, the prevalence and intensity in Catfish were higher than in Tilapia; this may be according to the feeding habits of Tilapia, which is herbivores and is less likely to get infected directly, while Catfish are omnivorous that will feed on all intermediate hosts (Malvestuto & Ogambo-Ongoma, 1978). These are compatible with the present study in that the highest prevalence and intensity were recorded in L. niloticus which represented 100%, followed by H. forskahlii, which have a prevalence of 82%, and the lowest were in O. niloticus and T. galilaea that recorded prevalence of 35.6 and 0.14% respectively.
Human anisakidosis (previously known as anisakiasis) is a disease that became of major health and economic importance. Humans were considered accidental hosts in the life cycle, as a result of consumption of raw fish, and these nematodes never develop inside the alimentary canal of human and may penetrate the tract and associated organs with severe pathological consequences. This disease was recorded in Egypt by Cocheton, Cabout, and Lecomte (1991). Early evisceration is recommended to avoid anisakidosis disease, due to the movement of the larvae from the digestive tract into muscles within a few days (Declerck, 1988). Also, freezing at − 20 °C for 3 days or heating to 70 °C because the larvae can survive at 50 °C. Sakanari and McKerrow (1989) indicated that larvae resist salting, smoking, and 51 days in vinegar. According to the US Food and Drugs Administration (FDA) agency, all fish products not intended for cooking or processing at temperature > 60 °C should be deep frozen at − 35 °C for > 15 h, or at − 23 °C for a minimum period of 7 days (Deardorff, Kayes, & Fukumura, 1991). Interestingly, C. magnipapillatum was redescribed from cormorants in Wadi Al-Raiyan lake area, Fayoum, Egypt, by Al-Bassel, 2006. In the present study, Hydrocynus forskahlii was the most Egyptian traditional salted fish (local name is Kalb-El Samak). This fish was exposed to direct sun in open air for about 24 h. The larger sized fish were gutted before being salted while small fishes were salted as a whole. Contracaecum larvae infected this fish by a prevalence of 82% and this may affect the human health in Egypt.
The SSCP analysis of ITS-1 and ITS-2 revealed five possible profiles for each. The sequence and phylogenetic analyses showed that the nematode larvae investigated in this study are closely related to C. multipapillatum, which is a species complex and it was shown that it consists of at least 4 distinct species found in the USA, Europe, and Australia (Nadler, D’Amelio, Fagerholm, Berland, & Paggi, 2000; Mattiucci, Turchetto, Bragantini, & Nascetti, 2002; D'Amelio et al., 2007; Shamsi et al., 2008).
It seems that Contracaecum larvae in Lake Nasser are belonging to C. multipapillatum complex and differ from each other in the sub-species level, with the possibility of the presence of at least one new species. It is necessary to complete the life cycles of different larvae of Contracaecum in the laboratory (running experiments) and examine adult worms using detailed morphological examination and molecular biology with complete ITS sequences and additional genetic markers.
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We would like to thank PD. Dr. Norbert Brattig and PD. Dr. Klaus Erttmann (Tropical Medicine Section, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) for providing their laboratory facilities during the preparatory experiment in this work.
There are no external sources of funding for this work except from the institutions to which they were affiliated.
The authors declare that they have no competing interests.
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