Sampling Techniques in Cetaceans

Tim Tristan DVM
Oso Creek Animal Hospital
Texas State Aquarium
Corpus Christi, Texas


Collection of various biological samples is the cornerstone of preventative medical care and early detection of disease processes in cetaceans. The advancement of training animals for medical behaviors has also made collection of many of these samples easier. The training implemented allows for routine collection of the samples on an “as needed” basis with a low level of stress to the animals. Groups such as IMATA (International Marine Animal Trainers Association) routinely promote and educate animal handlers on many of these routine medical behaviors. The following is a brief description of the techniques implemented for collection of common biological samples from cetaceans.


Gastric sampling
The gastrointestinal system of cetaceans is complex in its anatomical structure, but similar to many terrestrial animals. Diagnostics that are commonly performed on gastric samples include cytology, culture and sensitivity, and pH. The results from these tests may prompt treatment for gastric ulcers, parasites, or bacterial and fungal infections. In addition, further diagnostics may be needed such as radiography, ultrasonography, or endoscopy based on the results. Sample collection usually yields the best results if the stomach is empty of food contents.
Collection of a gastric sample may be performed with a variety of gastric tubes commonly used in equine medicine. Foal gastric tubes may be used in smaller animals (i.e. juvenile bottlenose dolphins (Tursiops truncatus)), while larger animals may require adult equine nasogastric tubes (i.e. Pilot whales (Globicephala macrorhynchus), adult bottlenose dolphins (Tursiops truncatus)). The length of the tube must be appropriate to reach the stomach. Premeasuring the tube from the tip of the rostrum to the level of the dorsal fin is a rough estimate of the length that is needed to reach the stomach in bottlenose dolphins (Tursiops truncatus). Marking the tube prior to the procedure allows identification of the length necessary to access the stomach. Passage of the tube is similar to advancing a nasogastric tube in a horse. The tube is advanced to the level of the blowhole and rotated 90 degrees. The tube is then passed caudally until the mark on the gastric tube is reached or “gurgling” of stomach contents is heard through the tube. The tube is then crimped and slowly withdrawn using smooth, fluid traction. Once the sample is identified in the tube, a sterile sample container is used for sample storage until evaluation can be accomplished.
The sample should be evaluated for color, consistency, odor, and pH prior to preparation for cytology or culture and sensitivity. The normal pH from a fasted dolphin ranges from 1.5-3.0. Contamination with seawater may affect these results and lower the pH causing erythrocyte lyses.

Cytology
Cytology is one of the most frequently implemented diagnostics used for evaluation of the gastrointestinal system. Wet mount samples are examined first, followed by samples stained with Dif-Quik or other comparable staining solutions. Many of the cells noted on microscopic examination originate from the oral mucosa, larynx, esophagus, stomach, and the respiratory tract. Cells commonly found are epithelial cells, leukocytes, erythrocytes, histiocytes, macrophages, protozoa, fungi, and parasite eggs. Candida albicans, Aspergillosis spp., and Cunninghamella spp. are a few of the more common fungal pathogens that can be found on cytology. Three types of epithelial cells are seen in gastric samples. Squamous epithelial cells are seen from the esophagus and fundic stomach, columnar epithelial cells are seen from the second stomach, and basal cells are seen from necrosis in the first and second stomach. Normal reference values are listed below.

Cell type (#/hpf)

Epithelial cells* _ greater than 5
WBC* _ 0-5
RBC _ 0
Histiocytes/macrophages _ 0-2

*As a general rule of thumb, there should be 2-3 times as many epithelial cells as white blood cells. If the ratio reaches or exceeds 1:1 it is considered abnormal.

Parasites are occasionally seen in captive dolphins but are more frequently seen in wild dolphins and dolphins in rehabilitation. Occasionally food fish are infested with adult and larval nematodes, reiterating the necessity for a sample collection from a fasted dolphin with an empty stomach. A few of the parasites seen in gastric samples are listed below.

Nematodes
Anisakis, Contracaecum, Pseudoterranova, Halocercus

Trematodes
Nasitrema, Braunina

Findings on cytology that should be considered significant include an increase in the ratio of white blood cells to epithelial cells, red blood cells, basal cells from the gastric mucosa, yeast, and fungi. Some marine mammal experts consider some parasites such as Nasitrema to be nonpathogenic, but they do have the capability of causing clinical illness.

Culture and Sensitivity
C&S is sometimes as difficult to interpret in cetaceans as it is in their terrestrial counterparts. Deciding whether an organism is pathogenic or commensal is not always an easy determination. Combined with physical exam, clinical signs, and other diagnostics, culture and sensitivity may be more useful. Bacterial organisms that may be pathogenic include Pseudomonas spp., Aeromonas spp., Clostridium and many others. The difficulty in interpreting this organisms pathogenicity is complicated by the fact that they naturally occur in the waters where the animals are housed. Fungal organisms that are seen include Candida albicans, Cunninghamella, and Aspergillus. Canidida is a frequent pathogen contributing to gastritis and digestive abnormalities. Candida may be found on gastric cytology and is considered normal in small numbers and lack of clinical signs in the patient.


Respiratory sampling
The respiratory system of cetaceans is complex and has special adaptations for animals with an entirely aquatic lifestyle. One of the more notable anatomical variations is the larynx. The larynx crosses through the esophagus and enters the palate, ventral to the bony nares. The tip of the larynx is held in place by a sphincter resulting in complete separation of the air and food passages. Between the bony nares and the blowhole is a complex series of valves, air sacs, tubular sacs, and musculature that is beyond the scope of this lecture. The point is, this particular adaptation may significantly affect the samples that are submitted to the laboratory and may not completely represent disease processes occurring in the lower respiratory system.
Diagnostics commonly performed include cytology and culture and sensitivity. Results from these tests may prompt the clinician to treat for pneumonia, parasites, and a variety of other illnesses.
Collection of samples from the respiratory system can be acquired by various methods. Captive animals may be trained to exhale into a sterile sample container or a petri dish. Samples from untrained animals may be collected by using sterile culturettes inserted directly into the blowhole when opened and removed when the blowhole opens again. Collection from untrained animals may also be accomplished with sterile sample containers or a petri dish if strong or productive exhales or “chuffs” can be anticipated or elicited by animal caretakers. Increased possibility of contamination by environmental organisms during sampling procedures also must be taken into account when interpreting results. Other sampling techniques, like bronchoaveolar lavage, may be accomplished with patients under sedation and bronchoscopy.
Similar to gastric samples, respiratory samples should be evaluated for color, consistency, and odor. Many times particular bacteria and yeast have familiar odors that may aid in initial treatment protocols until culture results can be obtained.

Cytology
Cytology is the most frequently utilized diagnostics in regard to respiratory sample examination. Wet mounts are examined first, followed by stained samples. Cells noted on microscopic exam originate from the nasal sacs, pterygoid sinus, larynx, bronchi, trachea, and lungs. Organisms consist of bacteria, yeast, fungal hyphae, and parasites. Normal reference values are listed below.

Cell type (#/hpf)

Epithelial cells* _ greater than 5
WBC* _ 0-5
RBC _ 0
Protozoa _ 0-1

*As a general rule of thumb, there should be 2-3 times as many epithelial cells as white blood cells. If the ratio reaches or exceeds 1:1 it is considered abnormal.

Parasites are frequently seen in captive dolphins in open ocean environments, wild dolphins, and dolphins in rehabilitation settings. A few of the parasites seen on cytology are listed below.

Nematodes
Halocercus, Pharurus, Pseudalius, Stenurus

Trematodes
Hunterotrema, Nasitrema

Protozoans
Kyaroikeus cetarius, Jarrellia atramenti

Findings on cytology that should be considered significant include an increase ratio of WBC’s to epithelial cells, presence of macrophages or band neutrophils, erythrocytes, presence of fungal hyphae, and an increased number of budding yeast.
As stated before, some parasites such as Nasitrema and Kyaroikeus are considered nonpathogenic in most cetaceans, but do have the potential to cause disease in compromised animals.

Culture and Sensitivity
C&S of respiratory samples, like gastric samples, are sometimes difficult to interpret. Combining physical examination, clinical signs, hematology and biochemical results aids the clinician in forming a more complete evaluation of the patient in question. Bacterial organisms that have the potential to cause disease include, but are not limited to, Pseudomonas, Aeromonas, Klebsiella, etc. Fungal organisms include, but are not limited to, Asperillus, Candida, and Cunninghamella. Candida in small numbers is considered normal.


Fecal sampling
Fecal samples are collected with a sterile polyethylene tube passed in to the anal orifice to the intestinal tract. These sterile, flexible tubes measure 0.5cm in diameter and 1.27 meters in length. The tube is passed up to 40cm into the intestines to obtain a fecal sample. Negative pressure is usually not necessary as the liquid fecal matter flows into the tube unassisted. Aspiration may alter the sample cellularity by traumatizing the intestinal mucosa. Visual evaluation of the sample should include color, consistency, and presence of mucus or blood.

Cytology
Cytological evaluation of fecal samples is indicated when gastrointestinal signs are noted, when abnormal feces is seen, or for routine evaluation. Wet mount samples are examined, followed by fecal flotation, and stained fecal smears. Cells noted on cytology may originate from the anus, intestine, duodenum, stomach, or respiratory tract. Cells commonly encountered include epithelial cells, white blood cells, degenerate white blood cells, red blood cells, protozoa, fungi, and parasite eggs. Squamous epithelial cells originate from the anus while columnar epithelial cells originate from the intestines and rectum. Intact clumps of epithelial cells are occasionally noted with rough sample collection with the fecal tube or with intestinal ulceration and erosion. Normal reference values are listed below.

Cell type (#/hpf)

Epithelial cells _ 0 - TNTC
Intact WBC _ 0-5
Degenerate WBC _ 0-10
RBC _ 0-3


Parasites may also be identified on fecal cytology and some are more commonly found on fecal cytology than gastric cytology. Gastric and fecal examination allows for a more complete picture of the animals digestive tract and aids the clinician in formulating a treatment protocol. A few of the parasites seen in fecal samples are listed below.

Nematodes
Halocercus, Anisakis, Contracaecum, Psuedoterranova

Cestodes
Eight genera, Strobilocephalus triangularis

Trematodes
Ten genera, Braunina, Nasitrema, Campula rochebruni

Acanthocephalans
Bolbosoma spp.

Two of the parasites listed above deserve further comment. Strobilocephalus triangularis is the only cestode considered pathogenic because it penetrates the colon wall and forms necrotic ulcers. The second, Campula rochebruni, may be of clinical relevance because of its effects on the liver, bile duct, and pancreas.

Findings on cytology that should be considered significant include an increase in degenerate WBC’s, increase in WBC’s and RBC’s, yeast with hyphae formation, fungi, one morphological type of bacteria, Clostridial organisms, and parasites. As stated earlier, some parasites are considered nonpathogenic.

Culture and Sensitivity

See Culture and Sensitivity for gastric samples.


Blood sampling
One of the most important samples collected in captive and rehabilitation cetaceans is blood. The anatomical structure of the cetacean peripheral vascular system decreases the available sites for venipuncture and increases the difficulty in sample collection. In addition, the vessels accessed in the fluke and flippers are perarterial venous retia containing mixed venous and arterial blood. The most frequently utilized site for venipuncture is the fluke. Other sites include the dorsal fin, pectoral flipper, and peduncle. Collection from smaller animals may require ¾-1.0 inch 18-22 gauge needle or butterfly, while larger animals require 1.0-1.5 inch, 18-22 gauge needle or butterfly. Both methods may utilize a vacutainer set up to aid in sample collection. The caudal peduncle requires a 1.5-3.5 inch, 18-20 gauge needle for sample collection.

Interpretation of hematology and biochemical samples will be discussed in the following lecture.
Tim Tristan, D.V.M.

Timothy E. Tristan received his DVM from Texas A&M University in 1999. From 1999 through 2001, Dr. Tristan practiced as an associate veterinarian at Marathon Veterinary Hospital while being a volunteer for Marathon Sea Turtle Hospital, Marathon Wild Bird Center and Key West Wild Bird Center. He also was a staff veterinarian for the Dolphin Research Center in Grassy Key, Florida. From 2001 to 2002 Dr. Tristan was an Avian and Exotic animal intern at Gulf Coast Veterinary Specialists in Houston, TX where he also was a relief veterinarian at the Animal Emergency Clinic in Houston and a contract veterinarian at Moody Gardens in Galveston, TX. Today, Dr. Tristan is an associate veterinarian at Oso Creek Animal Hospital in Corpus Christi, Texas, a contract veterinarian for Landry's Downtown Aquarium in Houston, Texas, Senior Staff Veterinarian for the Texas State Aquarium in Corpus Christi and a volunteer veterinarian for the Animal Rehabilitation Keep in Port Aransas, Texas and the Texas Marine Mammal Stranding Network for the Texas Gulf Coast.
Dr. Tristan holds professional licenses and memberships in the American Veterinary Medical Association, Texas Veterinary Medical Association, Association of Avian Veterinarians, Association of Reptile and Amphibian Veterinarians, and the International Association of Aquatic Animal Medicine.