Acute myocarditis in a West Indian Manatee , Trichechus manatus ( Sirenia : Trichechidae ) , from Puerto Rico

A 12-year old, adult male, West indian Manatee, Trichechus manatus, which stranded in Ponce, Puerto Rico 26 August 1992, died from cardiac failure. Suppurative epicarditis, myocarditis and endocarditis with mild to moderate degeneration and necrosis was caused by an unidentified infectious agent. Moderate to heavy parasite respiratory infections with Cochleotrema cochleotrema and intestinal infections with Chiorchis groschafti and stomach infections with Heterochelius tunicatus probably contributed to the physiological compromise of the host’s condition. This case represents the first record of a diagnosed natural mortality in the Antillean Manatee from the Caribbean and the first attributed to cardiac failure. Rev. Biol. Trop. 56 (Suppl. 1): 277-283. Epub 2008 May 30.

The West indian Manatee, Trichechus manatus Linnaeus is an herbivorous marine mammal found in coastal marine and fresh water and is an endangered species throughout its range in the Caribbean.Manatee mortality assessment has been documented in Florida since 1975.Studies on manatee mortality in Florida have been successful in documenting causes of death, identifying diseases and genetic conditions affecting the species, monitoring the impact of debris and pollution on manatee mortality, describing the parasite fauna affecting the species, analyzing watercraft-related mortality and propeller wounds as a cause of death, and age and reproductive biology (O' Shea et al. 1985, 1991, Ackerman et al. 1995, Wright et al. 1991, 1995).Although, the manatee is the most common marine mammal found dead in Puerto Rico, comprising 40.9% of all marine mammal mortality records (Mignucci-Giannoni 1990, Mignucci-Giannoni et al. 2000), little is known about the factors causing natural mortality.Between 1990 and 1995, the average number of death cases per year was six, increasing an average of 8.3% each year (Mignucci-Giannoni 1990, Mignucci-Giannoni et al. 2000).We describe the gross and histopathological findings of a necropsy of a stranded, wild West indian Manatee that succumbed to cardiac failure.Mignucci-Giannoni et al. (2000) mentions this mortality, but did not describe it.

MATERiALS AND METHODS
An adult West indian Manatee was reported stranded by a local fisherman in Barrio Mercedita, Sector La Esperanza, Ponce, Puerto Rico (17˚59.0'N,66˚35.4'W) on the morning of 26 August 1992.The animal was rolling in the subtidal shore attempting to breath, but was reported to have died soon afterwards.The case was designated CSN catalogue number NEPST194 and transported to the Caribbean Stranding Network (CSN) necropsy facility on Magueyes island off La Parguera, Puerto Rico, at the Magueyes island Laboratories of the Department of Marine Sciences, University of Puerto Rico at Mayagüez, and a complete necropsy was preformed on the fresh carcass following the protocols of Bonde et al. (1983) and Geraci and Lounsbury (1993).Digenea were fixed and preserved in steaming 5% formalin, stained in Harris' hematoxylin, dehydrated, cleared in beech-wood creosote, mounted in Canada balsam, and identified with Beck and Forrester (1988) and Yamaguti (1971).Nematodes were relaxed in glacial acetic acid, preserved in 70% ethanol, examined in glycerin, and identified with Sprent (1980) and Yamaguti (1961).Parasites were deposited in the U.S. National Parasite Collection, Beltsville, Maryland (USNPC).Tissue samples of the heart, lungs, liver, and intestine were collected and preserved in 10% neutral-buffered formalin at a fluid to tissue ratio of 10:1 for histological examination.Tissue samples were prepared in 5 μm paraffin sections stained in hematoxylin and eosin, Giemsa, Gram stain, or periodic acid-Schiff.Heart sections were also stained with bacterial-specific stains (Brown-Bren and Brown-Hopps) (Luna 1968).Stomach contents were rinsed in tap water over a 30-mesh (0.52 mm) screen to remove dirt and other fine particles that hamper microscopic examination.Stomach samples were preserved in 5% formalin (Bonde et al. 1983) and prepared and examined following the methods of Hurst and Beck (1988) and Mignucci-Giannoni and Beck (1998).A modified microscope point technique (Hurst and Beck 1988) was employed to quantify the relative proportions of stomach contents.The complete skeleton was preserved, cleaned, and deposited in the University of Puerto Rico (Rio Piedras) Biology Museum (UPR BM MM0C042) (Mignucci-Giannoni et al. 1997).

RESULTS
The animal had no external lesions or significant scarring.Both flippers were freely movable and normal in appearance.Total length (straight line, snout to fluke) of the animal was 258 cm, maximum girth 170 cm, and weight 245 kg.The animal was estimated to be approximately 12 years of age based on 12 growth layer groups in one of the ear bones from the skull (Marmontel 1993).An adequate nutritional status was indicated by the ventral outer and inner blubber thickness (32 and 20 cm, respectively).Stomach contents (67.0%unidentifiable rhizomes, 12.0% Halodule sp., 14.2% Thalassia testudinum, 6.4% Syringedium filiforme) were normal for this species in Puerto Rico with the exception of a slightly smaller percentage of T. testudinum (Mignucci-Giannoni and Beck 1998).
Moderate numbers of the opisthotrematid trematode, Cochleotrema cochleotrema (USNPC 78642) were found in the nares, larynx, trachea, bronchi, and bronchioles.Both lungs had generalized congestion.Eighty percent of the anterior mucosa of the colon was covered with the paramphistid trematode, Chiorchis groschafti (USNPC 78643).A heavy mucoid layer intermixed with the ascarid nematode, Heterochelius tunicatus (USNPC 78641) covered the mucosa at the duodenal diverticulum and anterior jejunum.Several nematodes were also free in the gastric lumen.
The most significant gross pathological findings were the presence of approximately 2 l of yellow transudate in the peritoneal cavity and variably sized pale areas of necrosis throughout the myocardium.The pericardial sac contained approximately 100 ml of clear transudate.Numerous ecchymotic and petechial hemorrhagic foci were present in the mediastinum, pericardium, epicardium, and over the serosal surface of the small intestines (Fig. 1).
Microscopically, the myocardial interstitium was diffusely infiltrated with abundant heterophils and minimal lymphoid cells that effaced myocardial fibers.Many of the myocardial fibers had numerous, closely spaced vacuolations throughout, while others were markedly eosinophilic and homogenous with loss of the characteristic cross striations.The pulmonary interstitium was diffusely congested with occasional alveolar spaces containing an eosinophilic, homogenous material consistent with a proteinaceous edema.There was generalized hepatocellular vacuolations-fat/glycogen. A few hepatocytes had an intracytoplasmic brown-green, grandular material, possibly bile.Kupffer's cells were engorged with a similar material and the surrounding sinusoids contained small numbers of heterophils.There was perivascular hemorrhage and proteinaceus edema fluid mixed with numerous heterophils and a small number of lymphoid cells in the epicardial layer.Similar inflammation extended into the endocardium.

DiSCUSSiON
The most serious lesions were in the heart.Suppurative epicarditis, myocarditis, and endocarditis with mild to moderate degeneration and necrosis were present (Fig. 5).No specific infectious agent was identified with the use of special stains; however, bacterial or other infectious agents were suspected because of the abundance of inflammatory cells and other pathological characters of the lesions (Fig. 2-5).
Little is known about infectious agents in wild West indian Manatees and specific diagnostic tests have not been developed (Wetzold     et al. 1994).The gross and microscopic abnormalities in the heart were sufficiently extensive to attribute death to heart failure.The accumulation of fluids was probably secondary to heart failure.The moderate to severe parasitism probably added to the physiological stress induced upon the animal by the probable infectious agent.The respiratory parasite, C. cochleotrema, has been reported to occur commonly in the manatee from Florida (Beck andForrester 1988), Puerto Rico (Mignucci-Giannoni et al. 1999a) and the Dominican Republic (Mignucci-Giannoni et al. 1999b), and Chiorchis spp.are abundant in manatees worldwide (Sprent 1980, Beck andForrester 1988), including Puerto Rico (Mignucci-Giannoni et al. 1999a) and the Dominican Republic (Mignucci-Giannoni et al. 1999b).Sweat and Wright (1994) reported valvular endocarditis in a stranded, wild, 16 to 17 year old, pregnant adult female Florida Manatee.Their description of the microbiological conditions associated with their case was limited due to the remote site and inability to collect culture samples.They concluded that the disease condition was caused by an unidentified bacterial infection, and suggested the added stress of pregnancy killed the female.Since the animal was not completely fresh, no examination for associated parasites, which seemed to play a role in our case, was possible.Similar heartvalve problems have been reported from manatees in Florida with some frequency (Buergelt et al. 1984, 1990, S.D. Wright, Fla. Mar. Res. inst., pers. comm., 1996).Cold Stress Syndrome can cause myocardial degeneration in manatees in Florida, but many other disease factors are also involved (Bossart et al. 2003).Heart disease in wild West indian Manatees may be more common than previous records would suggest.
Many carcasses of manatees are too decomposed to determination the cause of death (Bonde et al. 1983, Buergelt et al. 1984, O'Shea et al. 1985, Mignucci-Giannoni et al. 2000), and in Puerto Rico 33.7% of the manatee cases were obtained in a state of moderate to advanced decomposition (Mignucci-Giannoni et al. 2000).
This case is the first natural mortality diagnosed in the West indian Manatee in the Caribbean and the first attributable to cardiac failure in Puerto Rico and the Caribbean.

ACKNOWLEDGMENTS
Manatee carcass salvage and specimen collection were conducted under U.S. Fish and Wildlife Service (USFWS) permits PRT 2-8430 and PRT -684532 and under yearly permits from and a cooperative agreement with the Puerto Rican Department of Natural and Environmental Resources.Partial support for this study was provided in grants from the Caribbean Field Office, USFWS, and Save the Manatee Club.We thank Cathy A. Beck, Sirenia Project, U.S. Geological Survey, for identifying and quantifying the stomach contents, and confirming our parasite identifications; M. Marmontel, Project Marimua, Brazil, for assistance in aging the manatee; CSN volunteers for general assistance; and R.K. Bonde, Sirenia Project, and S.D. Wright, Florida Department of Environmental Protection, for comments on the manuscript.

Fig. 1 .
Fig. 1.Ventrodorsal aspect of the heart of a West indian Manatee (# NEPST194) showing numerous echymotic and petechial hemorrhagic foci of the epicardium and myocardium.Maximum length of the organ is 24.2 cm.

Fig. 3 .
Fig. 3. Photomicrograph of a section of epicardium and subjacent myocardium from a West indian Manatee (# NEPST194) with suppurative myocarditis, epicarditis, and endocarditis.Notice the expansion of the epicardium with edema and inflammatory cells (large arrow) and a few congested blood vessels (arrowheads).Hematoxylin and eosin stain.Bar = 550 μm.

Fig. 4 .
Fig. 4. Photomicrograph of a section of epicardium and subjacent myocardium from a West indian Manatee (# NEPST194) with suppurative myocarditis, epicarditis, and endocarditis.Notice the dense accumulation of heterophils within the epicardium (large solid arrows) and hyalinization of subjacent myocardial fibers (large open arrow).Hematoxylin and eosin stain.Bar = 28 μm.

Fig. 5 .
Fig. 5. Photomicrograph of a section of myocardial lesion from a West indian Manatee (# NEPST194) with suppurative myocarditis, epicarditis, and endocarditis.Notice the accumulation of interstitial areas with edema (arrowhead) and inflammatory cells (large arrow).Hematoxylin and eosin stain.Bar = 550 μm.