Molecular and morphological evidence of Taenia omissa in pumas ( Puma concolor ) in the Peruvian Highlands

A total of 41 cestodes were collected during necropsy examination on 2 pumas ( Puma concolor ) that were found in 2 communities in Canchis province, Cuzco region, Peru, at 4500 meters above sea level (Peruvian Andes). The cestodes were evaluated morphologically and molecularly. A fragment of the mitochondrial cytochrome c oxidase subunit 1 gene ( cox1 ) was used as a genetic marker. All the cestodes were identified as Taenia omissa . In the present report, we give a brief description by molecular and morphological diagnosis of the cestodes and compare nucleotide sequences with previous isolates from GenBank. Upon comparison, the sequences showed a difference in the cox1 gene of 5.1 to 5.3% with other teniids sequences. This finding constitutes the first report of T. omissa in Peru and expands the geographic distribution of this parasite

The present study confirms morphologically and molecularly the occurrences of T. omissa parasitizing two adult male pumas in Cuzco, Peru.This finding represents the first report of T. omissa in Peru.
In May and September of 2013, 2 adult male pumas were found dead, apparently killed by poachers, on two farms that were operated as alpaca production systems, located in the Pumaconca (14°18ʼ51.73ʼʼS,71°09ʼ07.41ʼʼW)and Abra La Raya (14°28ʼ43.03ʼʼS,71°01ʼ41.34ʼʼW)communities, in the province of Canchis, Cuzco region in Peru, at 4500 meters above sea level.The carcasses of the two pumas were donated for necropsy by the Technical Administration for Forestry and Wildlife of Peru to the veterinary research center IVITA-Marangani, at the Universidad Nacional Mayor de San Marcos.During the necropsy, twenty-eight and thirteen complete cestodes were collected from the small intestines, respectively.The parasites were fixed in 4% formaldehyde and then preserved in 70% ethanol.Some gravid proglottids were preserved in absolute ethanol for molecular studies.
Scoleces were mounted in Berlese's medium to facilitate observation and measurement of rostellar hooks.The rostellar hooks were measured in accordance with the parameters described by Haukisalmi et al. (2011).Both mature and gravid proglottids were stained with Semichon's acetocarmine stain and were dehydrated in an ascending alcohol series up to absolute ethanol.Subsequently, the samples were cleared in clove oil and terpineol, and then mounted in Canada balsam.
Photographs were taken using a Carl Zeiss microscope (Axioskop 40).Measurements were made using image analysis software (Leica IM50, version 4.0 R117).The measurements are reported in micrometers unless otherwise stated.Measured characteristics are given as range, with averages and standard error (SE) values in parentheses.The parasite taxonomic nomenclature used in this study follows Loos-Frank (2000) and Rausch (1994).The host taxonomic nomenclature follows Currier (1983).
Total DNA was extracted from three tapeworms from the puma from Pumaconca (To1, To2 and To3) using Chelex100, in accordance with the methodology described by Gadau (2009) with a minor modification.Tissue samples from gravid proglottids of approximately 1-3 mm 3 were put into 0.2 mL plastic vials and were kept at 37 °C for 30 minutes.The tissue was then completely dried, and 100 µl of 5% Chelex solution and 1 µl of proteinase K (20 mg/mL) were added into the vials.The vials were then incubated in a thermocycler using the following program: 1 hour at 57 °C, 10 minutes at 95 °C, 1 minute at 37 °C, 10 minutes at 95 °C, and finally 15 minutes at 15 °C.The DNA samples were then stored at -70 °C until use.
PCR was used to amplify an approximately 400-bp fragment of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) using the primers JB3 and JB4.5 (BOWLES & MCMANUS, 1994).The PCR solution was prepared in a 50 µl volume containing 4 µl of template DNA, 0.25 µM of each primer and GoTaq  Green Master Mix, 2X (Promega, Madison, WI, USA).A three-step thermal process consisting of 94 °C for 30 seconds, 55 °C for 30 seconds and 72 °C for 30 seconds was repeated 36 times to amplify the short fragment of cox1 (LIU et al., 2011).The PCR products were analyzed by means of electrophoresis on 1.5% agarose gel with ethidium bromide staining.The PCR products were then sequenced using the Big Dye TM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) and an ABI 3100 automated sequencer (Applied Biosystems).The sequences were assembled using the ChromasPro 1.7.6 software (TECHNELYSIUM, 2015).All the sequences were compared with a reference sequence in GenBank using ClustalX (CLUSTAL, 2015).A phylogenetic tree was constructed by means of the neighbor-joining method with the Kimura two-parameter distance, using the MEGA6 software (MEGA, 2015) (TAMURA et al., 2004;TAMURA et al., 2013).Unique nucleotide sequences of the partial cox1 gene of T. omissa from these pumas were deposited in the GenBank database under accession numbers KR095312, KR095313 and KR095314.
All the cestodes studied (41) were identified as T. omissa.The puma from Pumaconca was infected with a total of 28 cestodes (11 mature and 17 immature tapeworms).The puma from Abra La Raya was infected with a total of 13 tapeworms (9 mature and 4 immature tapeworms).
Nucleotide sequences of the cox1 gene were generated for PCR-positive isolates from three T. omissa (To1, To2 and To3).The first two were from the Pumaconca puma, and the third was from the Abra La Raya puma.The genetic identity of T. omissa was calculated from the alignments of the nucleotide sequences of the cox1 gene (Figure 2 and 3).Sequences of T. omissa from this study (GenBank Nos.KR095312 (To1), KR095313 (To2) and KR095314 (To3)) had single nucleotide differences of 0.7% between each other.However, the sequences for the To1 and To3 isolates were identical.All the sequences were compared with a sequence previously published for this species of tapeworm that had been collected from a puma in Canada (GenBank No. JX860631) (LAVIKAINEN et al., 2013).The isolate from Canada differed with regard to cox1 by 5.1 -5.3% from the sequences in this study.
Nucleotide sequences of the cox1 gene showed intraspecific variation of 0.7-5.3% between all the T. omissa isolates, including the isolate from Canada (JX860631).Similar variations have been demonstrated in other taeniid cestodes.Lavikainen et al. (2008) performed molecular phylogeny on various taeniid cestodes and  found an intraspecific variation of 0.0-3.4% in Taenia mustelae, 0.0-6.8% in Taenia polyacantha and 1.3-9.8% in H. taeniaeformis (LAVIKAINEN et al., 2008).The life cycle of T. omissa has been described by Forrester & Rausch (1990), they identified metacestodes collected from white-tailed deer (Odocoileus virginianus) in southern Florida, and morphological analysis showed the metacestodes to be T. omissa cysticerci (FORRESTER & RAUSCH, 1990).Currently, two species of deer are known to be involved in the life cycle of T. omissa, i.e.Odocoileus hemionus and O. virginianus (JENSEN et al., 1982;FORRESTER & RAUSCH, 1990;PYBUS, 1990).In Peru, O. virginianus has wide geographic distribution that includes the Andes mountain range and overlaps with the geographic distribution of the puma (SMITH, 1991).Therefore, we predict that O. virginianus is involved in the life cycle of T. omissa in Peru.Futures research will be needed to identify the animal species involved as an intermediate host in the life cycle of T. omissa in the Peruvian highlands.

Table 1 .
Haukisalmi et al. (2011)nia omissa (in µm) isolated from pumas in the Andes mountain range of Peru.Selected parameters were based on the system used byHaukisalmi et al. (2011).