Evaluation of coelioscopic splenic biopsy and cloacoscopic bursa of fabricius biopsy techniques in pigeons (Columba livia).

Abstract: Techniques for endoscopic biopsy of the spleen and bursa of Fabricius were evaluated in 71 juvenile and adult white Carneau pigeons (Columba livia). Splenic biopsy was performed under isoflurane anesthesia by a left lateral approach to the coelom from behind the last rib. From within the left abdominal air sac, the spleen was located just caudal to the isthmus of the proventriculus and ventriculus and ventral to the gonad in 86% of pigeons examined. In the remaining 14%, extensive reproductive activity within mature female pigeons prevented visualization of the spleen. Splenic biopsy samples were successfully obtained from all birds in which the spleen could be found, and all were determined to be of good to excellent histologic quality. Endoscopic biopsy of the bursa of Fabricius was accomplished by cloacoscopy and saline irrigation with the pigeons in dorsal recumbency. The bursa of Fabricius was located and a biopsy was done in 28 (39%) of 71 pigeons. Initial histopathologic evaluation indicated that only 30% of the biopsy samples possessed lymphoid tissue of diagnostic quality. Splenic biopsy via coelioscopy appeared safe and rewarding in pigeons. Cloacoscopy provided an excellent view of the cloaca and appeared to be well tolerated; however, biopsy of the bursa of Fabricius is not recommended because of poor diagnostic value of the specimens and the risk of cloacal perforation.

Key words: endoscopy, biopsy, lymphoid tissue, spleen, bursa of Fabricius, avian, pigeon, Columba livia

Introduction

Endoscopy has proved to be a useful diagnostic tool in veterinary medicine, and few taxa have reaped the benefits more than Aves. (1-3) Rigid telescopes are routinely used in avian practice to evaluate the respiratory tract, gastrointestinal tract, and coelomic viscera, particularly the urogenital tract, liver, and kidneys. Most endoscopic systems use a rigid telescope housed within an operating sheath through which basic instruments can be inserted into the field of view. Endoscopy allows tissue and organ visualization and biopsy while being minimally invasive. (2,3) Despite the widespread use of rigid endoscopy, no studies have evaluated the safety of the procedure or the quality of biopsy samples obtained in any avian species. In this paper, we report the evaluation of a commonly used rigid endoscopic system for collecting splenic and bursal biopsy samples from white Carneau pigeons (Columba livia), discuss patient safety issues, and assess diagnostic specimen quality.

Materials and Methods

Study population

Seventy-one juvenile and adult white Carneau pigeons were used to evaluate endoscopic biopsy of the spleen and bursa of Fabricius. The flock was maintained in a free-standing enclosed loft that was temperature controlled to remain between 13[degrees]C and 27[degrees]C (55[degrees]F-80[degrees]F). Windows in the building offered a natural photoperiod. A commercial pigeon diet (Purina Mills, St Louis, MO, USA) and water were offered ad libitum. All aspects of this study were critically evaluated and approved by the University of Georgia Institutional Animal Care and Use Committee (AUP A2004-10186-0).

Endoscopic equipment

Four separate operating stations were similarly equipped for the endoscopic procedures (Table 1). Each station consisted of a 2.7-mm 30[degrees] Hopkins telescope, xenon light source, fiber-optic cable, endovideo camera, and monitor (Karl Storz Veterinary Endoscopy America Inc, Goleta, CA, USA) (Fig 1). In addition, 2 stations possessed digital still/video documentation equipment (Sony Image Capture Device and Digital Photo Printer, 9525-P, Karl Storz Veterinary Endoscopy America Inc). Telescopes, operating sheaths, and all instruments were cleaned with a neutral pH enzymatic cleaner (Renuzyme Plus, Getinge USA Inc, Rochester, NY, USA), sterilized by submersion in 2% glutaraldehyde (Cidex 14 day, Advanced Sterilization Products, Irvine, CA, USA) for 15 minutes, rinsed with sterile distilled water and placed on a sterile paper drape. This cleaning and sterilization procedure was repeated between every bird.

[FIGURE 1 OMITTED]

Anesthesia

A standard anesthetic and surgical procedure was followed for all pigeons. Before induction of anesthesia, the pigeons were fasted for 10-12 hours overnight and physically examined. Each bird was premedicated with butorphanol (1 mg/kg IM; Torbugesic Fort Dodge, Fort Dodge, IA, USA) 20 minutes before induction of anesthesia. Anesthesia was induced with 5% isoflurane (IsoFlo, Abbott Laboratories, North Chicago, IL, USA) in oxygen with an over-the-head mask connected to a modified Norman elbow nonrebreathing system (Bickford PC-2B, Bickford Inc, Whales Center, NY, USA). Each bird was intubated with a 2.5-mm or 3-mm ID uncuffed endotracheal tube with a Murphy eye (Riisch Inc, Duluth, GA, USA) and transferred to the operating room for endoscopy. Anesthesia was maintained by connection to a pressure-limited mechanical ventilator (Small Animal Ventilator, Bioanalytical Systems Inc, West Lafayette, IN, USA) with isoflurane in oxygen to produce a surgical depth of anesthesia.

Extensive anesthetic monitoring was undertaken. (4) Arterial catheterization was performed with a 24-gauge x 19-mm Teflon catheter (Terumo Surflo, 0.67 x 19 mm, 17 ml/min, Terumo Medical Corporation, Somerset, NJ, USA) in the ulnar artery at the distal end of the ulna or in the lateral dorsal metatarsal artery. Direct blood pressure was measured with a pressure tranducer (TruWave disposable pressure transducer, Edwards Lifesciences LLC, Irvine, CA, USA) linked to a patient monitor (90367 Ultraview 1030 monitor, SpaceLabs Medical Inc, Redmond, WA, USA) and zeroed to the atmospheric pressure at the level of the right atrium. Heart rate was obtained from the arterial line. A mainstream capnograph with a neonatal sensor (C[O.sub.2]SMO capnograph and Capnostat mainstream C[O.sub.2] sensor, Novametrix Medical Systems Inc, Wallingford, CT, USA) was used to record end tidal C[O.sub.2] partial pressure and respiratory rate. Temperature was also recorded with an oral probe linked to the patient monitor and maintained with electrical warming blankets. Arterial blood gases were analyzed with an arterial blood sampler with tip cap (PICO30, Radiometer Medical A/S, Copenhagen, Denmark).

Coelioscopy and splenic biopsy procedure

Each bird was positioned in right lateral recumbency. The wings were secured dorsad over the back, and the left pelvic limb was secured craniad to the neck with 2.5-cm self-adhering bandage (1-in Vetrap, 3M, St Paul, MN, USA). After removing few, if any, feathers, the left flank was aseptically prepared with a chlorhexidine scrub solution (Hibiclens, AstraZeneca Pharmaceuticals, Wilmington, DE, USA) and a single final wipe of 70% alcohol. A skin incision was made with a No. 15 scalpel blade immediately behind the last rib as it crosses the ventral border of the flexor cruris medialis muscle and continued caudad for 3-4 mm. Small hemostats were then used to bluntly dissect through the body wall into the caudal thoracic air sac. The sheathed telescope was then inserted through the surgical site into the caudal thoracic air sac and advanced caudad to penetrate the left abdominal air sac (Fig 2). To locate the spleen, the telescope was advanced craniomedial to the gonad and ventral to the isthmus of the proventriculus and ventriculus. Once the spleen was identified, 5-Fr biopsy forceps (Karl Storz Veterinary Endoscopy America Inc) were introduced down the instrument channel of the operating sheath to appear within the center of the endoscopic field of view. The telescope-sheath-forceps were advanced as a single unit onto the spleen, and a biopsy sample was collected. After withdrawing the biopsy forceps, the tissue sample was gently rolled onto a sterile cotton-tipped applicator moistened with sterile saline and transferred to a biopsy cassette. The procedure was repeated to obtain a second splenic biopsy sample before the cassette was closed and placed in 10% neutral buffered formalin. The skin was closed with a single suture of absorbable monofilament polydioxanone (4-0 PDS II, Ethicon, Somerville, NJ, USA).

[FIGURE 2 OMITTED]

Cloacoscopy and bursa of Fabricius biopsy procedure

The bird was moved into dorsal recumbency with the vent facing the surgeon. An intravenous administration set (10 drops/ml, Baxter Corp, Deerfield, IL, USA) was connected to each of the 2 irrigation ports of the operating sheath. The ingress administration set was connected to a 1 L bag of sterile saline warmed to 39.4[degrees]C (103[degrees]F) and suspended above the surgery table. The end of the egress administration set was placed in a bucket under the table. The administration sets and sheath were primed with sterile saline before inserting the sheathed telescope through the vent and into the cloaca. The vent was pinched around the sheathed telescope by using thumb and forefinger to maintain a seal as saline was permitted to flow into the cloaca and create an endoscopic working space (Fig 3). The flow of saline entering and leaving the cloaca was controlled by adjusting the sheath ports between the on and off positions. Starting from the coprodeum, the telescope was slowly withdrawn, and once the urodeal fold was visualized, the dorsal urodeal wall was examined for the bursa of Fabricius. If located, 5-Fr biopsy forceps were used to collect 2 biopsy samples from the bursa. Tissue samples were placed in biopsy cassettes and fixed in 10% neutral buffered formalin as previously described. After completing the endoscopic procedure, the pigeons were permitted to recover quietly in a heated cage and then were returned to their aviary. Neither postoperative antibiotics nor additional analgesics were provided. Birds were observed daily for any complications related to the biopsy procedure.

[FIGURE 3 OMITTED]

Histopathology

Splenic and bursal biopsy samples were routinely processed through graded alcohols to xylene, embedded in paraffin, sectioned at 4 gm, stained with hematoxylin and eosin, cover-slipped, and examined microscopically. Tissue architecture, cellular preservation, and diagnostic quality of the biopsy samples were scored as poor, good, or excellent.

Results

All 71 pigeons were clinically healthy with a mean weight of 406 [+ or -] 42 g before surgery. The anesthetic protocol was effective and safe. Entry into the left caudal thoracic and abdominal air sacs was straightforward and uncomplicated in all patients. The image quality of the coelomic viscera was excellent, and all structures could be easily identified. The spleen was located in 61 (86%) of 71 birds. In all birds in which the spleen could be visualized, biopsy samples were collected without complications (Fig 4). In 10 birds (14%) in which the spleen could not be visualized, each was a hen that was reproductively active. Large, pendulous ovaries, often in conjunction with hypertrophy of the oviduct and salphinx, obscured the spleen and rendered any biopsy procedure blind and unsafe. Mild to moderate hemorrhage occurred from almost every biopsy site; however, evidence of excessive uncontrolled hemorrhage was not observed, and hemostasis was achieved naturally within 1 minute. All splenic biopsy samples were determined to be of good to excellent diagnostic quality with good to excellent cellular preservation and tissue orientation.

[FIGURE 4 OMITTED]

Cloacoscopy with sterile saline irrigation provided an excellent view of the rectal orifice, coprodeum, urodeal fold, urodeum, ureteral, and, in females, oviductal openings (Fig 5). An obvious bursa of Fabricius was identified, and a biopsy was done in 28 (39%) of 71 pigeons. These birds were <1 year of age. One bird deteriorated clinically immediately after bursal biopsy and died. Necropsy revealed perforation of the bursa of Fabricius and flooding of the air sacs with irrigation saline. Of the 10 bursal samples that were submitted for histopathologic examination, only 3 (30%) possessed any lymphoid tissue of diagnostic quality. Therefore, further attempts at bursal biopsy were abandoned.

[FIGURE 5 OMITTED]

Discussion

Coelioscopy appears to be a safe and rewarding procedure for the examination of coelomic viscera in the pigeon and other avian species. (2,3) The 2.7-mm telescope, 14.5-Fr operating sheath, and 5-Fr biopsy forceps were effective for collecting splenic biopsy samples in this study and have been generally advocated for collecting other visceral tissue samples from birds. (2,3) The effects of coelioscopy and creating a direct connection between the air sacs and the external environment could complicate anesthesia. Respiration and the exchange of anesthetic gas via the telescope entry site can lead to anesthetic instability; however, the use of mechanical ventilation appears advantageous and is not associated with adverse cardiopulmonary changes. (4) Apart from preoperative butorphanol, further analgesics were not used in this study. However, the use of postoperative analgesics, including opiates and nonsteroidal anti-inflammatory drugs, should be routinely considered in clinical practice.

Neither perioperative nor immediate postoperative morbidity or mortality was associated with coelioscopy and biopsy. Hemorrhage after splenic biopsy, although common, was never considered clinically significant. Although not an issue in this study, postoperative coelomitis was considered a possible risk factor, and in clinical practice, manual cleaning of endoscopic instruments with enzymatic solutions combined with longer sterilization times of 20-30 minutes are recommended. (5-8) Sterile surgical drapes were not used during this study but would reduce surgical site contamination and are recommended in clinical practice. (9)

Sterile saline irrigation distends the cloaca and creates a working space essential for endoscopy. The use of controllable in- and out-flow enables feces and urates to be flushed from the cloaca to permit an unparalleled view of the cloacal structures, including the rectal orifice, coprodeum, urodeal fold, urodeum, and ureteral and (in females) oviductal openings. The bursa of Fabricius can be visualized in the dorsal wall of the urodeum just caudal to the urodeal fold. The bursa is large and patent in young birds but involutes in adults, making this an unreliable target structure for lymphoid biopsy. (10,11) Although the endoscopic biopsy technique is facilitated by saline irrigation, complications are fatal if the cloaca is perforated and the air sacs are flooded. Furthermore, obtaining a good quality diagnostic biopsy specimen of the bursa is difficult. Therefore, although endoscopic biopsy of the bursa of Fabricius cannot be recommended, cloacoscopy offers a practical means of diagnosing and treating cloacal disease, including biopsy and diode laser ablation of cloacal papillomas and adenocarcinomas, and is recommended as a diagnostic technique in birds. (2)

The most substantial limitation to successful soft tissue biopsy involving the coelom is the relatively small size of most avian patients and the limited surgical access afforded by standard coeliotomy techniques. (12) Both of these limitations can be largely overcome by endoscopy, which provides focal magnification, illumination, and surgical access within the coelom. Surgeons performing surgery on humans have indicated that considerable benefits might be gained from minimally invasive laparoscopy, including rapid and accurate disease diagnosis, reduced need for extensive laparotomy, reduced surgical stress, improved postoperative pulmonary function, reduced hypoxemia, reduced surgical time, and faster recovery. (13-18) Significant morbidity has not been demonstrated in humans when subjected to appropriate laparoscopic techniques. (17) The efficacy, complications, or long-term effects of endoscopy have not been documented extensively in birds, although studies at the University of Georgia continue to critically evaluate these methodologies. Since this initial study, a total of 217 pigeons have been subjected to >400 coelioscopic splenic biopsy procedures with face mask anesthesia and minimal monitoring, and only 2 additional deaths have been recorded. This study demonstrates the value of minimally invasive endoscopy in birds for obtaining tissue samples for research studies as well as for diagnosing clinical disease. The 2.7-mm telescope system has proved to be both safe and effective and is recommended as a valuable tool for the avian researcher and practitioner.

Acknowledgments.. We thank the students and technical staff of the College of Veterinary Medicine for their support and assistance and Mr Kip Carter of Educational Resources for preparing Figures 2 and 3. We also thank Karl Storz Veterinary Endoscopy America Inc for supporting the research and development program at the University of Georgia. This study was part of a larger research project investigating pigeon circovirus funded by The National Aviary.

References

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(2.) Hernandez-Divers S J, Hernandez-Divers SM. Avian diagnostic endoscopy. Comp Cont Educ Pract Vet. 2004;26:839-852.

(3.) Taylor M. Endoscopic examination and biopsy techniques. In: Ritchie BW, Harrison G J, Harrison LR, eds. Avian Medicine: Principles and Application. Lake Worth, FL: Wingers Publishing; 1994: 327-354.

(4.) Touzot-Jourde G, Hernandez-Divers S J, Trim CM. Cardiopulmonary effects of controlled versus spontaneous ventilation in pigeons anesthetized for coelioscopy. J Am Vet Med Assoc. 2005;227:1424-1428.

(5.) Ayliffe G. Decontamination of minimally invasive surgical endoscopes and accessories. J Hosp Infect. 2000;45:263-277.

(6.) Cronmiller JR, Nelson DK, Salman G, et al. Antimicrobial efficacy of endoscopic disinfection procedures: a controlled, multifactorial investigation. Gastrointest Endosc. 1999;50:152-158.

(7.) Herruzo-Cabrera R, Gil-Miguel A, Fernandez-Arjona M, Rey-Calero J. Efficacy comparison of glutaraldehyde-phenate vs other glutaraldehydes in fomites disinfection, by different methods. Minerva Med. 1994;85:563-568.

(8.) Jette LP, Ringuette L, Ishak M, et al. Evaluation of three glutaraldehyde-based disinfectants used in endoscopy. J Hosp Infect. 1995;30:295-303.

(9.) Rutala WA, Weber DJ. A review of single-use and reusable gowns and drapes in health care. Infect Control Hosp Epidemiol. 2001;22:248-57.

(10.) Ciriaco E, Muglia U, Germana G. An ultrastructural study of pigeon bursa of Fabricius during involution. Anat Anz. 1989; 169:67-73.

(11.) Ciriaco E, Pinera PP, Diaz-Esnal B, Laura R. Age-related changes in the avian primary lymphoid organs (thymus and bursa of Fabricius). Microsc Res Tech. 2003;62:482-487.

(12.) Bennett RA, Harrison GJ. Soft tissue surgery. In: Ritchie BW, Harrison G J, Harrison LR, eds. Avian Medicine: Principles and Application. Lake Worth, FL: Wingers Publishing; 1994:1096-1136.

(13.) Kehlet H. Surgical stress response: does endoscopic surgery confer an advantage? Worm J Surg. 1999;23:801-807.

(14.) Corson SL, Grochmal SA. Contact laser laparoscopy has distinct advantages over alternatives. Clin Laser Mon. 1990;8:7-9.

(15.) Golditch IM. Laparoscopy: advances and advantages. Fertil Steril. 1971;22:306-310.

(16.) Lagares-Garcia JA, Bansidhar B, Moore RA. Benefits of laparoscopy in middle-aged patients. Surg Endosc. 2003;17:68-72.

(17.) Vander Velpen GC, Shimi SM, Cuschieri A. Diagnostic yield and management benefit of laparoscopy: a prospective audit. Gut. 1994;35: 1617-1621.

(18.) Yu SY, Chiu JH, Loong CC, et al. Diagnostic laparoscopy: indication and benefit. Zhonghua Yi Xue Za Zhi (Taipei). 1997;59:158-163.

From the Department of Small Animal Medicine and Surgery (S. J. Hernandez-Divers, Wilson, Lester, S. M. Hernandez-Divers, Ritchie) and the Department of Pathology (Latimer), College of Veterinary Medicine, University of Georgia, Athens, GA 30602-7390, USA.

Table 1. Endoscopic equipment used for coelioscopic
biopsy of the spleen and bursa of Fabricius.

Hopkins telescope, 2.7 mm x 18 cm, 30[degrees] (64018BSA (a))
Taylor operating sheath, 14.5 Er, 5-Fr instrument
  channel (67065CV (a))
Biopsy forceps, 5 Fr (69117Z (a))
Nova xenon light source, 175 watts (201315-20 (a))
Light guide cable, 3.5 mm x 230 cm (495NA (a))
Veterinary video camera II ([69235106 (a))
Sony image capture device and digital photo printer
  (9525-P (a))
Medical-grade monitor (9213-B (a))

(a) Karl Storz equipment identification numbers.