Occurrence of endoparasites in wild Antillean manatees (Trichechus manatus manatus) in Colombia.

The recognized impact of parasites in wildlife populations demands surveillance of endangered species like the Antillean manatees (Trichechus manatus manatus) in Colombia. We conducted a parasitological survey in four rescued sea cows in order to document the parasite diversity of this sirenian in the Caribbean wetland of Colombia and contribute to the molecular characterization of its trematodes. The flukes Chiorchis fabaceus, Nudacotyle undicola and the protozoans Eimeria manatus and E. nodulosa were identified in analysed faecal samples. For C. fabaceus and N. undicola, partial regions of ribosomal RNA genes were amplified and sequenced in order to infer their phylogenetic relations. The current study constitutes a new sirenian host (T. manatus manatus) record for the genus Eimeria and the trematode N. undicola.

Introduction

Given that the Antillean manatee (Trichechus manatus manatus) populations in Colombia are continuously decimated (Trujillo et al., 2013) it seems imperative to strengthen the conservation programs. Therefore, the manatee health status has to be monitored especially on the level of pathogenic infections. Concerning parasitic infections, earlier studies reported on several metazoans (Beck and Forrester, 1988, Mignucci-Giannoni et al., 1999, Bossart et al., 2012) and protozoans (Lainson et al., 1983, Upton et al., 1989, Borges et al., 2011, Borges et al., 2017, Bossart et al., 2012, Bando et al., 2014) in manatees. Most of these studies were conducted on captive manatees or on carcasses thereby hardly reflecting the actual status of healthy free-ranging animals (Beck and Forrester, 1988, Mignucci-Giannoni et al., 1999, Borges et al., 2016). Due to their diving activities, apnea capacity and the typical nature of their habitats (turbid rivers laden with tannins and muddy wetlands), it is difficult to observe and sample free-ranging manatee continental populations in South America. Furthermore, to date, the pathological significance of parasites in manatees and respective life cycles are almost unknown (Beck and Forrester, 1988) and parasitological studies have been limited to the morphological description and identification of eggs and adult specimens in faecal or tissue samples. Consequently, no studies are available on the molecular identification of manatee parasites. Moreover, reports on natural occurring Eimeria infections in manatees are limited and included merely three studies reporting on E. trichechi infections in the Amazonian manatee (T. inunguis) (Lainson et al., 1983) and on E. manatus and E. nodulosa infections in Florida manatees (T. manatus latirostris) (Upton et al., 1989, Bando et al., 2014). In regard to our knowledge there are only two reports on N. undicola infections, both in the Florida manatee (Dailey et al., 1988, Bando et al., 2014). Therefore, the current report constitutes the first description of Eimeria species and N. undicola in Antillean manatees and thus extents their geographical distribution to manatee populations in the Caribbean wetland from Colombia. This study also adds useful molecular information for further research seeking to extent the knowledge on pathological, ecological and epidemiological aspects of manatee trematode parasites.

Materials and methods

Faecal samples of four rescued Antillean manatees were collected after spontaneous defecation during routine clinical examinations within the OMACHA Foundation and the ‘Corporación Autónoma Regional de los Valles del Sinú y del San Jorge (CVS)’ program for rehabilitation and conservation of manatees in Santa Cruz de Lorica, Department of Cordoba, Colombia (9°13′25.79″ N; 75° 50′ 33.92″ W) (Fig. 1). The sampling procedure was in concordance with the Guidelines for the Treatment of Marine Mammals in Field Research of The Society for Marine Mammalogy.

Fig. 1

A) Clinical evaluation of a calf manatee as part of the conservation programme of the OMACHA Foundation in Colombia. B) Blood collection for health status evaluation. C) ‘Ciénaga Grande de Lorica’, Cordoba, flowers of water spinach (Ipomea aquatica), are a favorite manatee food.

The samples were submitted for coprological analyses including the standard sodium acetate acetic acid formalin (SAF) technique with ethyl acetate (Yang and Scholten, 1977) and a modified sedimentation technique. Samples were examined microscopically and illustrated via a digital camera. Additionally, carbol fuchsin-stained faecal smears were conducted (Heine, 1982) for the detection of Cryptosporidium spp. oocysts. Commercial coproantigen-ELISAs were also performed for the detection of Giardia and Cryptosporidium infections (ProSpecT®, Oxoid).

For further molecular analysis, eggs of the trematodes were isolated and subjected to an eggshell destruction process through five freezing and thawing cycles. Samples were treated with liquid nitrogen for 1 min and following submitted to a rapid temperature increase to 99 °C. Thereafter, DNA was extracted using the DNeasy Blood & Tissue Kit® (Qiagen, Germany). Partial ribosomal regions of the small subunit (SSU), the large subunit (LSU) and 5.8S were amplified using the following specific primers: WormA, NF1, 18S, WormB, (for the SSU), ZX-1, NC2, Plagi 28S-r1, D3A, D3B (for the LSU) and NC1 (for the 5.8 S) (Littlewood and Olson, 2001). The final PCR reaction volume consisted of 50 μL containing 5 μL DNA template, 0.5 μL of bovine serum albumin (BSA, 10 mg/mL Sigma-Aldrich), 1 μL of forward- and reverse-primer (10 pmol/μl) and 10 μl of 5x HOT FIREPol Blend Master Mix 7.5 mM MgCl2 (Solis BioDyne, Tartu, Estonia). Reactions were performed in a Veriti 96 thermocycler (Life Technologies, Darmstadt, Germany) using the following cycling conditions: 95 °C for 15 min (initial denaturation), followed by 40 cycles of 95 °C for 20 s (denaturation), 54 °C for 30 s (annealing) and 72 °C for 2 min 30 s. PCR amplicons were isolated from a preparative agarose gel using the HiYield Gel/PCR DNA Extraction Kit (Süd-Laborbedarf, Gauting, Germany) and thereafter cloned into pDrive vector (Qiagen, Hilden, Germany). Isolated recombinant plasmid DNA and PCR amplicons were bi-directionally sequenced by LGC Genomics (Berlin, Germany). The obtained DNA sequences of C. fabaceus and N. undicola have been submitted to GenBank under the accession numbers MF370224 and MF538578, respectively. For phylogenetic analysis, the D2-D3 28S rDNA region was used. A redundant dataset of sequences was chosen from the highest scoring BLAST results of GenBank which were posteriorly aligned by MUSCLE and used for a phylogenetic analysis using maximum parsimony and maximum likelihood methods with 1000 bootstraps by means of Mega 7 (Kumar et al., 1994).

Results

Overall, the coprological analyses revealed two metazoan and two protozoan parasite species. Among the latter, neither Cryptosporidium nor Giardia infections were detected. The metazoan species consisted of monoxenous manatee trematodes, C. fabaceus (Diesing, 1838) and N. undicola (Dailey et al., 1988). Illustrations of the respective trematode eggs are shown in Fig. 2. Morphological characterization of C. fabaceus eggs (n = 21) revealed a mean length of 162 μm (137–175 μm) and a width of 115 μm (103–131 μm), with an ovoid shape and a well-defined operculum at one pole. The eggs contained a light brown granular content surrounded by a thin eggshell, as typical for trematode species (Fig. 2 A). Nudacotyle undicola eggs (n = 18) had a mean size of 16.9 μm (15.01–20.13 μm) x 9.0 μm (8.26–10.33 μm), with an ovoid shape and characteristic elongated filaments on each pole whose bases were of equal widths; the content was granular and slightly diaphanous (see Fig. 2D).

Fig. 2

Illustrations of endoparasite stages identified in faecal samples of Antillean manatees (Trichechus manatus manatus) from Colombia: (A) Chiorchis fabaceus egg, (B) Eimeria manatus oocyst, (C) Eimeria nodulosa oocyst, (D) Nudacotyle undicola egg.

The protozoan stages found in this study corresponded to oocysts of two Eimeria species. The unsporulated oocysts from E. manatus (Upton et al., 1989) had a mean size of 9.51 μm (8.58–11.95) x 9.09 μm (7.11–11.31), were spheroidal, with a thin, translucent oocyst wall lacking any micropyle and enclosing a spherical sporoblast within circumplasm (Fig. 2 B). Eimeria nodulosa oocysts (Upton et al., 1989) had a mean size of 12.05 μm (6.81–15.92) x 10.93 μm (10.60–13.87 10.37 μm), were spheroidal or sub-spheroidal with characteristic knob-like protrusions on their oocyst wall surface (Fig. 2C).

The phylogenetic analysis was performed on a sequence dataset of the D2-D3 region of the 28S rRNA gene (C. fabaceus: 859 bp; N. undicola: 853 bp). The overall topology of the inferred phylogenetic tree (Fig. 3.) agreed well with the analysis of Olson et al. (2003). As expected, the assignment of the two trematode species to superfamilies in phylogenetic analysis corresponded to their morphological taxonomy classification. Thus, the genus Chiorchis grouped to the superfamily Paramphistomoidea and Nudacotyle to the Pronocephaloidea.

Fig. 3

The phylogenetic tree including the here identified trematodes Chiorchis fabaceus and Nudacotyle undicola shows that they belong to the superfamilies Paramphistomatoidea and Pronocephaloidea, respectively. Fasciola hepatica (Fasciolidae) was used as the outgroup taxon.

Discussion

In the current study, manatees were identified to be infected with the trematode C. fabaceus (Diesing, 1838). Indeed, the first ever report on trematode parasites in sirenians referred to this parasite species. However, to date there are no studies about the pathogenicity and the life cycle of this manatee trematode (Beck and Forrester, 1988). In this sense the present molecular characterization may aid future investigations to reveal the life cycle of this trematode and help to unveil its pathogenicity in manatees. It is worth noting that the closely related genus Paramphistomum (Fig. 3) is known as pathogenic for other terrestrial herbivores, in which infections can cause considerable morbidity and even mortality, especially in young animals (Tandon et al., 2014). In this regard, a mortality rate of up to 90% has also been reported in manatee calves (Mignucci-Giannoni et al., 2000). Nowadays collisions with watercraft are considered as one of the most important cause of manatee mortality (Ackerman et al., 1995). In this respect, it may be speculated that high intensity C. fabaceus infections may lead to disease induced weakening of animals that may indirectly contribute to collision-derived mortalities. Clinical signs of the closely related disease, acute paramphistomosis in other herbivores include diarrhoea, anorexia, loss of body condition, dehydration, cachexia, apathy and decreased perception (Tandon et al., 2014). Mortalities of unknown aetiology (which account up to 69%, Ackerman et al., 1995) were also reported to be associated to cold weather conditions in combination to the so-called cold stress syndrome (CSS) (Bossart et al., 2003). Thus, the combination of CSS with energy imbalances due to gastrointestinal chiorchiosis in manatees might have additional adverse effects on manatee's health condition as reported for other herbivorous mammals infected with the related Paramphistomum trematode (Tandon et al., 2014).

The intestinal trematode N. undicola (Dailey et al., 1988) was also identified. Eggs of N. undicola, which are characterized by their peculiar morphology (Fig. 2 D), have previously been reported from manatees in the USA (Bando et al., 2014). In contrast to Bando et al. (2014), we noted that these eggs sometimes had two filaments at each pole. This unique morphological feature has also been observed in Ogmogaster spp. eggs of stranded sei whales (Balaenoptera borealis) from Patagonia, Chile (C. Hermosilla, personal communication), a closely related trematode. It has been postulated for other closely related trematodes with similar egg morphologies (e. g. Notocotylus) that these thin filaments might facilitate the infection of gastropod intermediate hosts. For N. attenuates (Erkina, 1954), these thin egg-derived filaments were also proposed to support the adhesion/formation of egg clusters, which may then easier be shed than single eggs (Graczyk and Shiff, 1993). In case of N. undicola, we did not observe any egg clusters, but this might be due to the small sample size.

So far, only three species within the genus Eimeria have been described in manatees, i. e. E. trichechi in T. inunguis (Lainson et al., 1983), E. manatus and E. nodulosa in T. manatus latirostris (Upton et al., 1989). The small number of oocysts found is in agreement with the report of Lainson et al. (1983). In addition, the range of oocyst sizes of each Eimeria species is in line with previous reports (Upton et al., 1989, Bando et al., 2014). Unfortunately, faecal samples were fixed thereby hampering oocysts sporulation and evaluation of more accurate morphological characteristics of these species. In general, Eimeria infections are more frequently reported in younger herbivorous animals than in older ones, since homologous reinfections generally result in immunological protection (Hermosilla et al., 2012, Taubert et al., 2009). Given that E. manatus and E. nodulosa oocysts were exclusively found in young animals in this study, this might also apply to manatees (although the sample size was too small to fully support this hypothesis). As reported for terrestrial herbivorous mammals, pathogenicity of Eimeria infection significantly depends on the species, the inoculum size, the age of the host, the localization within the host and the type of endogenous meront/gamont stages (Hermosilla et al., 2012). However, Lainson et al. (1983) correlated Eimeria oocyst shedding in manatees with clinical signs of coccidiosis. Consequently, certain Eimeria species in calf/young manatees might result in clinical coccidiosis as described for various ruminant livestock animals (Ruiz et al., 2013).

Since hardly anything is known on intestinal parasites in free-ranging manatees in Colombia, the present study might serve as a baseline for future manatee monitoring health projects. It may also be useful for future research seeking to extent the knowledge on the trematode life cycles in manatees and to better understand their possible pathogenic implications for the health of Colombian wild manatee population.

Conflicts of interest

Declarations of interest: none.