Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2010172113 The bacTerial communiTy associaTed wiTh The leech Myzobdella lugubris leidy 1851 (hirudinea: Piscicolidae) from lake erie, michigan, usa Schulz c.* & FaiSal M.* Summary: Résumé : Communauté baCtérienne assoCiée à la sangsue myzobdella lugubris leidy 1851 (Hirudinea : PisCiColidae) du Leeches are widespread in the Great Lakes Basin, yet their potential laC érié, miCHigan, usa to harbor disease-causing agents has not been investigated. The purpose of this study was to identify the bacterial community of the Les sangsues sont répandues dans le bassin des Grands Lacs, commonly occurring leech, Myzobdella lugubris, within the Lake cependant, leur capacité d’héberger des agents pathogènes n’avait Erie Watershed. Leech samples were collected from the pectoral pas été investiguée. Le but de cette étude était d’identifier la fins of channel catfish, Ictalurus punctatus, and freshwater drum, communauté bactérienne de la sangsue commune, Myzobdella Aplodinotus grunniens, from Lake Erie in commercial trap nets and lugubris, du Lac Érié. Les échantillons de sangsue ont été collectés pooled into two samples based on host attachment. Bacteria from sur des poissons-chats, Ictalurus punctatus, et des poissons within the viscera of M. lugubris were identified by sequencing their perciformes, Aplodinotus grunniens. Les bactéries présentes dans les 16S rRNA (rDNA) gene of amplified community bacterial DNA viscères de M. lugubris ont été identifiées par le séquensage de leur extracted from pooled leech homogenate samples and were 16S rRNA (rDNA) et les échantillons ont été comparés à deux checked for similarity in two public databases: the Ribosomal bases de données publiques : Ribosomal Database Project et Database Project and BLAST. Bacteria belonging to the phylum BLAST. Des bactéries appartenant aux phyla Bacteroidetes, Bacteroidetes, β-proteobacteria, Verrucomicrobia, and unclassified ß-proteobacteria, Verrucomicrobia et d’autres bactéries non Bacteria were present in the leech samples. A large number of classifiées étaient présentes dans les échantillons de sangsues. bacteria found within leeches attached to channel catfish consisted De nombreuses bactéries trouvées dans des sangsues parasitant of sequences that could not be classified beyond the Domain le poisson-chat n’ont pas pu être classifiées au-delà du genre. Bacteria. However, many of these sequences were homologous Cependant, beaucoup étaient homologues (< 45 %) au phylum (< 45 %) to the phylum Bacteroidetes. One of the five genera Bacteroidetes. Un des cinq genres détectés dans l’ensemble des detected in the leech homogenates was Flavobacterium sangsues était Flavobacterium psychrophilum, une bactérie psychrophilum, a serious fish pathogen that causes Bacterial Cold pathogène responsable de la “Maladie des eaux froides” chez les Water Disease. While the occurrence of genera varies, bacteria poissons. Les bactéries associées à la sangsue étaient les mêmes associated with the two fish species were similar. pour les deux espèces de poisson mais d’occurrences variables. Mots clés : Key words: Myzobdella lugubris, leech, bacterial community, Lake Erie. Myzobdella lugubris, sangsue, communauté bactérienne, Lac Erié. INTRODUCTION haematophagous feeder, dominated the leech popula- tions in both lakes and heavily parasitized the channel catfish (Ictalurus punctatus) and freshwater drum eeches (hirudinea: Piscicolidae) are ubiquitous ( Aplodinotus grunniens). throughout North america (Klemm 1991; Muzzall let al. 1987; Klemm et al. 2003). Most recently we it is known that leeches can harbor potentially harmful identified leeches prevalent on lake St. clair and lake bacteria (Graf, 1999; Kikuchi & Fukatsu, 2005; laufer et Erie fish (Schulz et al., submitted, 2010). Epidemiological al., 2008). While there have been a few studies descri- studies demonstrated that leeches are more widespread bing bacteria within leeches used for medicinal purposes in lake St. clair than originally thought, with a preva- (Graf, 1999; Silver et al., 2007; laufer et al., 2008), to lence of 7.79 %, a mean intensity of 6.45 leeches/fish, the best of our knowledge, this is one of the first studies and an abundance of 0.50 leeches/fish (Schulz et al., describing bacterial communities of leeches which feed submitted, 2010). Myzobdella lugubris, an intermittent, exclusively on fish. The European medicinal leech, Hirudo spp., is of particular interest due its use in aiding venous congestion complications that occur following * aquatic animal health laboratory, Department of Pathobiology and surgical procedures in humans and its unique rela- Diagnostic investigation, Department of Fisheries and Wildlife, tionship with Aeromonas spp. (Sawyer, 1986; Silver et Michigan State university, S-112 Plant Biology, East lansing, Mi, 48824 al., 2007). Hirudo spp. contains multiple Aeromonas correspondence: M. Faisal Tel.: + 1 (517) 432-8259 – Fax + 1 (517) 432-8260 spp., such as A. veronii Biovar sobria, A. hydrophilia, E-mail: [email protected] and A. jandaei in its digestive tract (Graf, 1999; Silver Parasite, 2010, 17, 113-121 Mémoire 113 Schulz c. & FaiSal M. et al., 2007; laufer et al., 2008). Aeromonas jandaei is 5.1 cm stretch mesh, 7.6 cm stretch mesh hearts and also a symbiont in the digestive tract of the North ame- wings, and 91.4 m long leads of 10.2 cm stretch mesh. rican medicinal leech, Macrobdella decora (Siddall Fish were removed from the nets after 48 hrs and exa- et al., 2007). additionally, a recent study has shown that mined for the presence of leeches. The attachment sites the bladder of H. verbana harbors several species of were recorded for each fish and leeches were separated bacteria, including a Niabella spp., which is similar to into whirlpaks containing lake water according to the Flavobacterium johnsoniae (Kikuchi et al., 2009). infec- fish number and attachment site. leeches remained alive tions have also been known to occur in human post- until returned to the laboratory, where they were iden- operative patients, for which Hirudo spp. have been tified as Myzobdella lugubris based on the morpholo- used for bloodletting (Silver et al., 2007; laufer et al., gical key of Peckarsky et al. (1990). leeches, ranging 2008). in size from 7.5 mm to 20 mm, were disinfected by Pathogenic bacteria have also been found within the dipping them in absolute ethanol for 30 sec prior to digestive organs of leeches. Kikuchi & Fukatsu (2005) dissection (Graff, 1999). leech viscera were aseptically found a Rickettsia spp. infection in the frog leeches removed, with the assistance of a dissecting microscope, Torix tagoi and T. tukubana, and the fish leech Hemi- and placed in a microcentrifuge tube containing 80 % clepsis marginata. Rickettsia is a known pathogen that ethanol (Thermo Fisher Scientific, Pittsburgh, Pa). Due seriously affects many fish species (cusack et al., 2002). to the small size of the leeches, internal organs were also, leeches that fed on infected fish contained patho- homogenized and then pooled. Sequence analysis was genic strains of Aeromonas spp. and Pseudomonas spp. performed on eight leeches which were collected from in their digestive tracks (Snieszko & Bullock, 1968). the pectoral fins of channel catfish and four leeches additionally, Dombrowski (1952) demonstrated that which were collected also from the pectoral fins of Piscicola geometra transmits Pseudomonas punctata to freshwater drum. carp, which is known to cause moralities in farmed fish (altinok et al., 2006). leeches are also known to disperse sequenCe analysis A. hydrophila from host to host (Negele 1975). addi- tionally, Aeromonas spp. can cause lethal infections in Genomic bacterial community DNa was harvested from the leech homogenates (30-50 mg) using the Power- farmed and wild fish (harikrishnan & Balasundaram, TM 2005), as well as septicemia and diarrhea in humans Soil DNa isolation Kit (MO BiO laboratories inc., (Janda & abbott, 1998). To this end, we used 16S rRNa carlsbad, CA) following the manufacturer’s protocol. (rDNa) gene sequencing to identify bacterial commu- The PcR amplification of the bacterial 16S gene were nities associated with the internal organs of leeches. performed using the universal eubacterial primer set 27f-1387r (27f: 5’-aGaGTTTGaTc(AC)TGGcTCAG-3’ and 1387r (5’-GGG cGG WGT GTa CAA GGc-3’) (Marchesi et al., 1998). The PcR mixtures (25 µl/reac- MATERIAL AND METHODS tion) contained 20 pmol 27F and 1387R primers, 22 mM Tris-hcl (ph 8.4), 55 mM Kcl, 1.65 mM Mgcl2, 220 µM study area and fisH samPling dNTP’s, 0.55 units recombinant Taq DNa Polymerase, ish were collected from the western portion of and 50 ng template DNa (all reagents from invitrogen lake Erie, where Myzobdella lugubris are found life Technologies, carlsbad, ca, uSa unless otherwise to occur (41° 46’ 00.74” N, 83° 24’ 58.09” W, stated). The PcR amplification was carried out for F 30 cycles of 94 °c for 4 min., 56 °c for 30 sec. and 72 °c Fig. 1). lake Erie is the smallest and shallowest of all the Great lakes (Gl), and is located near the southeas- for 1.5 min. and final 7 min. incubation at 72 °c (modi- tern portion of Michigan. lake Erie supports a commer- fied after Sambrook and Russell 2001). cial fishery for a wide variety of fish, including the The expected size of PcR products was 1.36 kb. The channel catfish (Ictalurus punctatus), freshwater drum PcR products were used to construct 16S gene clone ® (Aplodinotus grunniens), bigmouth