Abstract

Effective diagnosis of suspected or confirmed disease in dolphins often presents many challenges to the clinician. Diagnostic imaging techniques including endoscopy, sonography, radiology, computed tomography (CT), magnetic resonance imaging (MRI) and thermography each have inherent indications and limitations. To address many of the limitations of these diagnostic techniques for the evaluation of thoracic disease, fine needle aspiration biopsy (FNAB) of intra and extra-thoracic structures in the dolphin has been investigated. This paper reports our clinical experience illustrated by findings in three cases.

Introduction

Imaging techniques often effectively localize pathology but do not give etiologic diagnoses. Respiratory endoscopy of dolphins is still within the developmental stage. Our experience has shown that collection of materials from airways via lavage or brush is often limited by the animals' physiologic status, risk associated with out-of-water time and general tolerance of the procedure. Furthermore, directing lavage or brush techniques to areas with lesions may be difficult. Lesions may also be located in lung regions not accessible via an airway approach. Percutaneous biopsy techniques have been described in marine mammals (Sweeney, 1990) however it appears they are only rarely employed in practice.

Fine needle aspiration biopsy (FNAB) techniques are easy, low-risk and provide aseptically collected samples directly from imaged lesions for cytological, microbiologic and chemical analyses. Results of these analyses often lead to valuable information for clinical decision making. FNAB is indicated for the further characterization of accessible lesions when other techniques do not provide a definitive diagnosis or carry too high a risk/benefit ratio.

The technique of FNAB is routinely used in other animal species and the methods of specimen collection, processing and interpretation are widely reported (Cowell and Tyler, 1989; Mills, 1989; Rakich and Latimer, 1989). Minimal risk is associated with needle aspiration biopsy techniques when a body cavity is not entered. Reported complications from percutaneous FNAB of lung lesions in other species include, pneumothorax, minor hemoptysis, hemothorax, subcutaneous emphysema and air embolism. All are infrequent, generally minor and usually require no treatment (Rakich and Latimer, 1989). We anticipate that the unique anatomy of the dolphin skin, pleura and lung parenchyma will result in lower complication rates in these species. Specifically, we expect that the prominent elastic and connective tissue elements, the thick pleura and highly specialized skin elements will effectively lower risk of pneumothorax, iatrogenic hemorrhage or subcutaneous emphysema. Complications due to iatrogenic trauma are avoided by knowledge of the sonographic intrathoracic anatomy.

Methods

The technique of thoracic FNAB in dolphins follows that described for other species with few modifications. If the thoracic cavity is not entered with the needle, the site is prepared as for a routine injection. If the needle will enter the thoracic cavity the site must be prepared as for surgery and aseptic technique is used. Longer needles than are routinely used for other animal species are usually required due to the thick skin of the dolphin. We use spinal needles with stylets for FNAB. The stylet is useful to avoid plugs of blubber that may be cut as the needle is advanced through the skin. A stab incision in the skin at the site of needle entry with a No. 11 scalpel blade is also helpful in reducing blubber plugs. A 20-cc syringe is generally sufficient to create necessary negative pressure during aspiration and allows for ease of handling by the operator.

Lesions outside the thoracic cavity and of sufficient size can be aspirated blindly. The clinician triangulates the insertion of the needle so that the tip will be within the desired area when advanced to the appropriate depth. Redirection of the needle is difficult due to the thickness of the skin so a route is chosen that is expected to produce a useful sample with careful advance and withdrawal only. Intrathoracic lesions require ultrasound guidance and the procedure is best performed by two operators. One maintains sonographic visualization of the lesion and the anatomy along the needle course while the other advances the needle and performs the aspiration. Ultrasound guidance localizes the lesion in three dimensions. Needle guides that attach to the ultrasound transducer have not been very helpful in our experience. Sites along the caudal border of the ribs are avoided to prevent trauma to the intercostal vessels and nerves. The needle tip is advanced to the level of the intercostal muscles and the triangulation confirmed. The needle is then advanced into the region of interest immediately after a breath. In most cases, sufficient samples can be collected between breaths so that the needle does not remain in the thoracic cavity during a breath. However, we have had on several occasions a needle within lung parenchyma during a breath without recognized complication.

Results

The experience of our first case presented, in spite of a non-productive aspirate, increased our confidence in the safety and value of the technique. A 14-year-old female Atlantic bottlenose dolphin (Tursiops truncatus) presented with signs suggestive of a respiratory tract infection. Radiographs demonstrated several foci of increased mixed interstitial pattern deep within the left lung. Transcutaneous sonography demonstrated a subpleural lesion in the caudal ventral left lung measuring approximately 4 x 2 x 2 cm. FNAB was performed due to the location of the lesion and the animal's temperament. An 18 gauge x 3 ½" spinal needle was advanced into the lesion under ultrasound guidance after aseptic preparation of the skin. The sonographic appearance of the lesion changed markedly as the needle entered. The borders became much less distinct and the lesion much harder to visualize. The procedure was aborted over concern that the change in appearance was due to rupture of an abscess or bulla. The animal remained stable throughout the procedure and after. Five days later sonography of the area showed no evidence of complications and the lesion was not seen on the sonograms. Seven days later the lesion was again evident on sonography but no signs of complication from the procedure ever developed and the animal continued to improve clinically. The dolphin has since recovered completely and no lesions are evident on radiography or sonography.

A second case illustrates the indication for aspiration of a lesion that did not appear to be accessible via airway approach. A 14-year-old male dolphin developed a firm, progressive swelling of the left dorso-lateral thorax that was not adequately characterized with plain radiography or sonography. The swelling appeared to be adjacent to and possibly associated with the dorsal aspect of a rib and had a mixed echogenic pattern suggesting a fluid filled center. The only contributing history was the recent observation of the animal passing a tapeworm segment. The skin overlying the swelling was aseptically prepared and an 18 gauge x 3 ½" spinal needle was inserted into the center of the lesion under ultrasound guided triangulation. A large volume of serosanginous clear fluid was collected. Cytological assessment of the fluid was consistent with an organizing hematoma and ruled out a parasitic cyst or abscess, which would have required more aggressive therapy. The lesion resolved without further intervention and the animal remains clinically normal to date.

Another case illustrates the value of FNAB in an animal that was deemed too compromised for bronchoalveolar lavage (BAL) and the value of the technique in determining an etiological diagnosis. An 18-year-old male dolphin had progressive refractory lung disease documented with sonography and radiography, but no etiological diagnosis. A lung lesion was localized with ultrasound and the overlying skin prepared aseptically. An 18 gauge x 3 ½ "needle was directed with ultrasound guided triangulation to the center of the lesion. Initial aspiration was non-productive. A syringe pre-loaded with 10 cc of sterile non-bacteriostatic saline was attached to the needle and ~ 5cc was infused. Immediate aspiration produced a return that when analyzed by cytocentrifugation revealed cryptococcal organisms. This animal ultimately died from cryptococcal pneumonia. Necropsy results did not reveal any evidence of immunosuppression or underlying disease and earlier FNAB with definitive diagnosis may have led to successful treatment.

Discussion

The technique of FNAB has proven valuable in the clinical care of dolphins and holds promise for additional applications. Our experience has shown the technique to be rapid, safe and produce valuable information for case management decisions. We have not experienced complications as a result of FNAB in any of the procedures conducted to date. There are several lymph nodes that should be accessible to FNAB techniques in dolphins, specifically, the superficial cervical lymph node complex and the marginal lymph node of the lung. These structures are reliably imaged with diagnostic ultrasound and are expected to yield aspirates that reflect disease processes in the anatomy that drains to them. Our initial work toward obtaining FNAB of these nodes has been hampered by difficulty stabilizing the nodes within surrounding tissues. There are also some large cardiovascular structures near the marginal nodes, namely the heart and internal thoracic vessels that increase the risk of complications but with continued development we hope to further adapt FNAB to lymph node aspiration.

Acknowledgements

T. Romano lab for time critical in-house cytocentrifugation of FNAB samples. W. G. Miller for photomicrographs.

References

1.  Cowell, R.K. and R.D. Tyler. 1989. Cytology of Cutaneous Lesions. In" Parry, B.W. (ed.) The Veterinary Clinics of North America Small Animal Practice. Volume 19, Number 4, Clinical Pathology" Part I. W.B. Saunders Company, Philadelphia. Pp 769-794.

2.  Mills, J.N. 1989. Lymph Node Cytology. In: Parry, B.W. (ed.) The Veterinary Clinics of North America Small Animal Practice. Volume 19, Number 4, Clinical Pathology: Part I. W.B. Saunders Company, Philadelphia. Pp 697-718.

3.  Rakich, P.M. and K.S. Latimer. 1989 Cytology of the Respiratory Tract. In" Parry, B.W. (ed.) The Veterinary Clinics of North America Small Animal Practice. Volume 19, Number 5, Clinical Pathology: Part II. W.B. Saunders Company, Philadelphia. Pp 823-850.

4.  Sweeney, J.C. 1990. Surgery in Marine Mammals. In. Dierauf, L.A. (ed.) Handbook of Marine Mammal Medicine" Health, Disease, and Rehabilitation. CRC Press, Boca Raton, FL. Pp. 215-234.