Burmese Python (Python molurus bivittatus) - Science Direct

-j

J. Comp. Path. 1997 Vol. l l7, 283-288

C F

SHORT PAPER

A N o v e l Mycoplasma sp. A s s o c i a t e d w i t h P r o l i f e r a t i v e T r a c h e i t i s a n d P n e u m o n i a in a B u r m e s e P y t h o n (Python molurus bivittatus) j . D . Penner, E. R. Jaeobson*, D. R. Brown~, H. P. Adams++ and C. L. Besch-Willifordw VeterinaryMedical Diagnostic Laboratory, University of Missouri, Columbia, MO, *Department of SmaU Animal Clinical Sciences and tDepartment of Pathobiology, College of Veterinary Medicine, University of Florida, GainesviUe, FL, ~Electron Microscope Laboratory, New Mexico State University, Las Cruces, NM, and w of VeterinaryPathobiology, University of Missouri, Columbia, MO, USA Summary Proliferative lymphocytic tracheitis and pneumonia were observed histologically in the respiratory tract of a captive Burmese python (Python molurus bivittatus). A mycoplasma species was isolated from the respiratory tissue. Polymerase chain reaction analysis of the 16S rRNA gene sequence of the isolate showed 0"90 similarity to Mycoplasma agassizii, an organism previously shown to cause respiratory disease in reptiles. Based on these findings, a novel Mycoplasma species was suspected to be the causative agent of respiratory disease in this snake. @ 1997 W.B. Saunders Company Limited

Mycoplasma sp. cause subclinical or clinical respiratory infection in a variety of animals (Dungworth, 1993). Mycoplasma agassizii is responsible for an upper respiratory tract disease (URTD) of desert tortoises (Brown et al., 1994), the pathology of which is characterized by lymphoplasmacytic rhinitis, with proliferative changes similar to those seen in mammals with mycoplasma infection (Jacobson et al., 1991; Brown et al., 1994). Proliferative lung lesions have been seen in pythons at the University of Florida, Gainesville (Jacobson, unpublished), and in Burmese pythons previously submitted for diagnostic necropsy to the Research Animal Diagnostic and Investigative Laboratory (RADIL) at the University of Missouri, Columbia. This report describes a case of lymphocytic proliferative tracheitis and pneumonia in a Burmese python and the isolation of a Mycoplasma sp. from respiratory tissues. An adult male Burmese python was one of four privately owned snakes. In an outbreak of pneumonia, two of the four snakes died. The remaining two were treated with antibiotics, but one of them, a male, failed to recover fully. Although showing almost normal activity, it refused to eat and breathed through its open mouth if handled. Bubbles of clear mucus were occasionally seen on the scales at the labial commissures. The snake was humanely destroyed when it began to lose condition 8 months after the original outbreak. The snake was approximately 12 feet long and weighed 10"9 kg. At necropsy, 0021-9975/97/070283 + 06 $12.00/0

9 1997 W.B. Saunders Company Limited

284

J.D.

P e n n e r e t al.

increased mucus was seen in the trachea and lungs, and the parenchyma of the cranial portion of the lungs was mildly thickened. The respiratory tract secretions were aseptically swabbed for routine aerobic microbiological examination. Small pieces of tissue from the distal part of the trachea and proximal part of the lung were collected aseptically for Mycop[asma isolation, and additional samples were placed in neutral buffered formalin for histopathological examination. Representative tissue samples from all other major organ systems were also collected for histological examination. Respiratory tract secretions cultured aerobically at 37~ on blood agar and MacConkey's agar failed to yield pathogenic bacteria. The tracheal and pulmonary tissue samples, which had been placed in chilled Frey's medium and frozen at - 7 0 ~ were packed in dry ice and sent from the University of Missouri to the University of Florida for mycoplasma isolation. On arrival, the tissue samples were minced in SP4 mycoplasma medium, supplemented with either glucose or arginine. The supernate was incubated aerobically at 30~ until mycoplasma growth was detected by acidification of the broth; subcultures were then made on solid SP4 medium supplemented with glucose. A Mycoplasma sp. was isolated from both tracheal and pulmonary tissue samples, producing small to medium-sized colonies with a cauliflower or wart-like appearance. Polymerase chain reaction (PCR) amplification of the 16S rRNA gene of the mycoplasma was pertbrmed on a crude cell digest of mid-log phase growth organisms. Amplification primers were complementary to terminal sequences highly conserved in members of the class Mollicutes (Robertson et al., 1993). The PCR products were purified by gel-filtration spin chromatography and quantified by spectrophotometry before automated fluorescent dideoxynucleotide sequence determination (Perkin-Elmer Applied Biosystems, Foster City, CA, USA; model 373A, version 1.2.1) withbut molecular cloning. A partial sequence of approximately 400 nucleotides, spanning the first to third variable regions of the gene (Neefs et al., 1990), was obtained by extension from the amplification primer. Previously published mollicute 16S rRNA gene nucleotide sequences were obtained from GenBank of the National Center for Biotechnology Information, National Institutes of Health (Devereux et al., 1984; Bilofsky and Burks, 1988). Nucleotide sequences were compared by means of the VAX computer version 1.4 of the basic local alignment search tool (Altschul et al., 1990). Mycoplasmas cultured from the trachea and lungs shared the same 16S rRNA gene nucleotide sequence, which was distinct from any sequence currently in GenBank. Pairwise nucleotide sequence similarity scores, computed with default gap and gap length weights, indicated that the mycoplasma isolate closely resembled M. agassizii (90% similarity) and mycoplasma strain H3110 (86% similarity). As already stated, M. agassizii is a known aetiological agent of U R T D of tortoises (Brown et al., 1994); mycoplasma strain H3110 has also been isolated from the respiratory tract of sick tortoises (Brown et al., 1995). Formalin-fixed tissues were processed by routine methods, sectioned, and stained with haematoxylin and eosin (HE). Additional lung and tracheal sections were stained by a modification of Gram's stain (Brown and Hopps,

New Mycoplasma

Fig. I.

sp. in a Python

285

(A) l,umiH~ll n~'~ticm