Summary

A necropsy was performed on a fifty-five pound male fresh-water dolphin. A description of necropsy techniques employed, unusual anatomical structures observed in this marine mammal, and significant pathology found, is discussed. Hemorrhagic lesions observed throughout the lymphatic system and other tissues, supports a diagnosis of generalized bacteremia, probably erysipelas.

Introduction

The subject of this necropsy was one of the prize exhibits of a commercial aquarium. This animal was one of two specimens of this specie captured from the Amazon River by an expedition from the Aquarium of Niagara Falls in October of 1965. Up until that time, there were none on exhibit in public aquaria anywhere in the world. One of the Sotalia died during a ten-day observation period at Vero Beach, Florida, presumably from shock. The Sotalia and another fresh-water delphinidin (Inia geoffrensis), which had been capmed on the same expedition were exhibited together in a 10,000 gallon exhibit tank. The inter-generic behavior of these two animals has been reported by the curator, Stephen H. Spotte.¹¹ The history of this animal which had been on exhibit for almost two years, was of little help. While the handlers had noticed a slight decrease in food consumption for a day or two prior to finding the animal dead in the tank, his behavior appeared normal. The necropsy was performed approximately 2-1/2 hours following the time when he was found. There was no evidence of post-mortem decomposition.

External Appearance

This 55 pound male fresh-water dolphin showed numerous scars from old skin wounds of a minor nature. There was no evidence of any significant trauma externally. The eyes were bright with a slight discharge from the left eye. There was no discharge from the blowhole. Although the outer layer of epidermis showed evidence of dehydration, the curator suggests that just the length of time it had been out of the water in delivery from Niagara Falls to our facility, was sufficient to start peeling the epidermis. There was no evidence of any discharge of external openings of either the urogenital or digestive systems. The overall length of the animal was 52-1/2". Throughout this report, comparisons will be made with the bottle-nose porpoise (Tursiops truncatus) with which most aquarists are more familiar.²

Primary Incision

Primary incision was made along the ventral midline from the center of the mandible bypassing the urogenital and the anal openings to the pubis. The average thickness of the subcuticular fat layer on the mid-line was approximately 2 centimeters. The color of the muscles on cut section was a uniform dark red, somewhat lighter than observed in the Tursiops. The wings of the hyoid bone extended out laterally to a point just about 4 centimeters anterior to the point of attachment of the pectoral fins. They afford a considerable degree of protection to the entire structure of the larynx. The anterior margin of the laryngeal cartilage is shaped almost like the prow of an ocean liner. A mucopurulent- exudate was found in the posterior nares and posterior pharynx.

The epiglottis and this prow-like projection on the laryngeal cartilage both relate to the shape of the posterior nares, so that they can effectively occlude that opening when submerged. A section of the posterior pharynx was taken for microscopic examination which showed infiltration of leucocytes evidencing an inflammatory process involving the submucous layer, plus a leucocytic infiltration of the glandular tissue of the salivary glands in the pharyngeal region. In some of these areas, the lymphocytic infiltration was so extensive as to completely destroy the normal morphological characteristics of the glands.

On initially opening the abdominal cavity, there was no evidence of any excess fluid or exudate. The overall impression seemed to suggest a slight congestion of the serous surfaces of the intestines. The external surface of the liver appeared slightly mottled in color, varying from a light tan to almost black, with some light streaks and white foci, which could represent scars from previous parasitic infestation. The liver weighed approximately two pounds, and had a mottled appearance. Upon incision into the main bile duct, vast numbers of mature trematode flukes were observed. While definitive studies of this parasite were not conducted, preliminary study suggests they are the same as the Zalaphotrema hepaticum found in marine mammals.

Gastro-Intestinal System

Esophagus - The esophagus contained a milky, slightly yellow-tinged exudate with some white particulate material in it, probably mucous. Microscopically, there was some desquamation of the mucous membrane with leucocytic infiltration in the submucous area.

Stomach - The cardiac stomach was devoid of ingesta, with the exception of the exudate. The deeply folded mucous membrane of the glandular stomach appears grossly hemorrhagic, but as noted by John Aldus Huxley back in the eighteenth century, every cetacean that he ever examined, showed this, so he wondered if this was, in fact, a physiological condition of this organ.⁴ Microscopic sections of the various parts of the four stomachs were remarkably negative, with the exception of a few apparent cysts deep in the crypts of the glandular stomach.¹⁰ However, these in no way resemble the parasite Braunia cordiformis⁷ reported by this author in 1966² and by Schry'ver, et al, this year,⁹ as seen in the Tursiops truncatus.

Intestines - The pylorus was patent, although the opening was only approximately 5 mm. in diameter. Some small petechial hemorrhages were observed in the mucous membrane of the duodenum, less than 1 millimeter in diameter. The gross appearance suggested a point of previous parasitic attachment, or possibly an early peptic ulcer. Microscopic examination of these lesions showed that they were in fact, ulcers; however, in the exudate outside of one such lesion, there were two structures which took the staining characteristics of the cell membrane of nematode ova. Their internal structure, if it was that, resembled in many ways, an overlying large phagocyte.

The remainder of the intestinal tract proved to be a single undifferentiated organ for its entire length with no recognizable diverticulum, such as a cecum. The entire intestinal tract was empty of ingesta, with the exception of some bile-stained mucous in the first third. There were no adult nematode parasites observed.

Liver - Multiple incisions into the liver offered no particular resistance in any area. The capsule stripped readily from the surface; the texture of the organ was slightly friable, suggesting a degenerative process.

Pancreas - The pancreatic lymph nodes were grossly hemorrhagic. There were some areas of gross hemorrhage visible on the surface of the pancreas, which is a rather compact organ, measuring approximately 7 cm. in its greatest diameter, by about 5 cm., with an overall thickness at its highest point of 4 cm.

The pancreatic duct appeared considerably larger than what we normally encountered in other species. Upon opening the pancreatic duct, we found it to be the location of some additional fluke-type trematode parasites. In sectioning the pancreas, we found flukes invading all of the major ducts, with related inflammatory processes and congestion. Although the pancreatic duct appeared to be as much as one centimeter in diameter in its cross-section, the actual orifice into the intestine was no greater than would be anticipated in an animal of this size.

Respiratory System

Upon initially opening the chest cavity, there was no evidence of excessive pleural fluid. There were no pleural adhesions, although there was a gross impression of slight congestion on the parietal pleura. The mediastinal lymph nodes were grossly hemorrhagic. Microscopic examination confirmed this gross observation.

On opening the trachea, a bloody foam was observed throughout the length of the trachea. Microscopic sections of the trachea, showed a hemorrhagic tracheitis. Sections through several different locations in the lung revealed progressive pneumonic changes involving infiltration of leucocytes and in some areas, frank hemorrhage. Anatomically, the same cartilaginous support of very small bronchioles required for the large tidal volume of respiration, which are found in other delphinids, were observed.¹⁰

Cardiovascular System

The pericardium stripped easily, and appeared to be normal. There were multiple focal areas of hemorrhage in the epicardium of all four chambers of the heart. There was no evidence of infarction. The hemorrhages noted in the epicardium extended into the myocardium to varying depths from one to four millimeters.

Microscopic examination of sections of the auricle also revealed hemorrhagic lesions between the endocardium and myocardium in several areas. The heart valves showed a vegetative endocarditis which microscopically included deposition of vast amounts of fibrin encasing numerous gram-positive rods. Major blood vessels showed no significant findings.

The spleen, which was an ovoid-shaped organ, was about 7 cm. in its greatest length, by 5 cm. in its greatest width. It had what appeared to be two or three lobulations on the margin. In its external appearance, the spleen showed some engorged major vessels under the capsule. The cut surface appeared normal on gross examination. The apparent lobulation was a true anatomic separation.

The grossly hemorrhagic lymph nodes found in the mesentery were similar to those found in the mediastinum, as well as in the supporting tissues around the stomach and pancreas.

Genitourinary System

Kidneys - The kidneys appeared to be lobulated to approximately the same extent as seen in the Tursiops. They measured approximately 11 cm. in length, and 7 cm. at the greatest width, with the average thickness about 4 cm. On cut surface the kidneys appeared to be normal on gross examination. However, histologically the kidneys show an extensive interstitial nephritis, characterized by lymphocytic infiltration and hemorrhage.

Ureters and Urinary Bladder - No Significant Findings.

Urinalysis of a sample collected directly from the urinary bladder showed pH 6, trace of protein, negative for glucose and ketones, few red and white blood cells, and a specific gravity of 1.0219. These findings are consistent with an interstitial nephritis.

Penis - The penis of the Sotalia, in the relative position where the bovine has a sigmoid flexure, seems to coil in a spiral pattern, making a full 360° circle when retracted.

Testicles - The testicles were in the abdominal cavity, and measured approximately 6 cm. in length, by a maximum diameter of 4 cm. On microscopic examination, the germinal epithelium appeared to be active, although this was a relatively young animal.

Adrenal Glands

The adrenal glands, measuring 3 cm. in length by 2 cm. in the greatest diameter, on cut surface showed what appeared to be a normal cortex with a grossly hemorrhagic medula. These findings were confirmed histologically.

Bacteriological Evaluation

Direct smears of blood from the heart were stained, utilizing both the Wright-stain and the Gram-staining techniques. Numerous gram-positive rod form organisms consistent with the morphology of Erysipelothrix sp. were observed on direct examination of the stained blood smears.

Unfortunately, the attempts to culture blood, stomach content, and the froth from the trachea were victims of a laboratory accident, and therefore we were deprived of the opportunity to make a fully-confirmed laboratory diagnosis.

Diagnoses

1. Acute infectious pharyngitis
2. Tracheo-bronchitis (infectious)
3. Bronchial pneumonia
4. Trematodiasis
5. Choledochitis (parasitic)
6. Pancreatitis (parasitic and congestive)
7. Generalized bacteremia with a hemorrhagic syndrome compatible with erysipelas infections.³

Immediate Cause of Death. The overall impression is that the respiratory and parasitic problems by themselves would not have been of sufficient severity to cause death. The pancreatitis caused by the heavy trematode infestation may have contributed to a stress situation which proved fatal when challenged by the fever and hemorrhagic syndrome of the bacteremia.

Discussion

On the assumption that this animal did, in fact, die of an erysipelas infection, the recommendation made to the curator of the Aquarium involved three different approaches to prevention and control of the disease, each of which is dependent upon the other two as part of a concerted preventive medicine program:

A. Immunizations

• Erysipelas - While previously we had vaccinated the Tursiops with the killed bacterin, which is commonly used for immunization of swine, we switched to the use of a modified live culture, avirulent erysipelas vaccine, on the recommendation of Dr. Ridgeway, who has been using it for two years, with no ill-effect.
• Clostridium - On the recommendation of Dr. Klontz of the Western Fish Disease Laboratory, we vaccinated all of the Tursiops, and the remaining Inia, against clostridium perfringens, using a dosage of 2 cc of the killed bacterin subcutaneously, approximately 6 cm. to the left of the base of the dorsal fin.⁶

B. Sanitation: Lacking the dilution factor and the biological systems which maintain ecological balance in nature, it is extremely important that the level of pathogenic micro-organisms be kept to a minimum in the tanks in which these animals are either maintained or exhibited. It is, therefore, recommended that chlorine levels be maintained at a bacteriostatic level with periodic peaks of greater concentration in order to purge the system and kill off any resistant organisms which may have established themselves.

C. Food Fish: Since the most obvious source for the introduction of erysipelas, clostridia or any other pathogenic organisms is in the food fish,^5,8 every effort must be maintained to minimize the numbers of micro-organisms introduced from this source. The proper control of the food fish from the bacterial standpoint will, at the same time, provide protection against the development of excessive concentrations of histamine through the decomposition of protein, which serves as a further source of digestive problems in these animals, and may in fact, be the cause of the gastric ulcers which we have seen.¹The proper management of the food fish has several significant factors, most of which involve discipline of the staff, and will require adequate supervision for proper implementation as follows:

• Quality of Fish Purchased - All food fish purchased should be fit for human consumption of the best quality available, purchased in the frozen state.
• Thawing - Food fish should be thawed in a refrigerator at approximately 40°F. More specifically, thawing at either room temperature or by use of warm water should be avoided as either of these methods allows the outer surface areas and outer fish to reach temperatures considerably above 40° F. before the center of a large block gets thawed.
• Preparation for Feeding - Whether fish are to be fed whole or cut up, once they are prepared, they should be maintained under refrigeration at 40°F. until they are immediately used. Here discipline is required to avoid such practices as leaving buckets of food on the show platform between shows or between feeding periods. If necessary, for more efficient operations, thought should be given to the placing of a small refrigerator backstage.

Carrying out all of the above recommendations as fully as possible can in no way guarantee against the recurrence of infections of this type. However, they are a move in the right direction.

References

1. Geraci, J.R., and Gerstmann, K.E.: Relationship of Dietary Histamine to Gastric Ulcers in the Dolphin, JAVMA, Vol. 149, No. 7, P. 884-890, October 1, 1966.

2. Grafton, T.S.: Necropsy of a Post-Partum Dolphin (Tursiops truncatus), Abstract - 17th Annual Meeting of the Animal Care Panel, Chicago, Illinois, 1966.

3. Hagan, W.A., and Bruner, D.W.: The Infectious Diseases of Domestic Animals, 4th Ed. Comstock Publishing Associates, P. 167-179, 1961.

4. Huxley, T.A.: Anatomy of Vertebrated Animals, D. Appleton & Co., London P. 346-349, 1886-.--

5. Jasmin. A.M., and Baucom, J.: Erysipelothrix insidiosa Infections in the Caiman (Caiman crocidismus) and the American Crocodile (Crocidismusacutus), Am. J. Vet . Clin. Path. Vol. 1, No. 4. P. 173-177, December 1967.

6. Klontz, G.W., Personal Communication to Stephen H. Spotte, October 1967.

7. McIntosh, A.: New Host and Distribution Records for the Trematoda Genus Braunina Heide:, 1900, Journal of Parasitology, 39, P. 31, 1953.

8. Reichenbach-Klinke, H., Elkan, E.: The Principal Diseases of Lower Vertebrates, Academic Press, P. 190, 1965.

9. Schryver, H.F., Medway, W., and Williams, J.F.: The Stomach Fluke (Braunina cordiformis) in the Atlantic Bottle-Nose Dolphin, JAVMA, Vol. 151, No. 7, P. 884-886, October 1, 1967.

10. Slijper, E.J.: Whales, Basic Books, Inc., 1962.

11. Spotte, S.H.: Intergeneric Behavior Between Captive Amazon River Dolphins Inia and Sotalia, Underwater Naturalist, P. 9-13, Fall 1967.