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Kiricephalus coarctatus in an Eastern Indigo Snake (Drymarchon couperi); endoscopic removal, identification, and phylogeny. a
a
b
A. Paige Brock , Alexander E. Gallagher , Heather D. Stockdale Walden , Jennifer c
c
c
a
L. Owen , Mark D. Dunbar , Heather L. Wamsley , Amber B. Schoeller , April L. a
Childress & James F.X. Wellehan Jr
a
a
Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA b
Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA c
Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA Accepted author version posted online: 24 Jul 2012. Version of record first published: 02 Aug 2012
To cite this article: A. Paige Brock, Alexander E. Gallagher, Heather D. Stockdale Walden, Jennifer L. Owen, Mark D. Dunbar, Heather L. Wamsley, Amber B. Schoeller, April L. Childress & James F.X. Wellehan Jr (2012): Kiricephalus coarctatus in an Eastern Indigo Snake (Drymarchon couperi); endoscopic removal, identification, and phylogeny., Veterinary Quarterly, DOI:10.1080/01652176.2012.709952 To link to this article: http://dx.doi.org/10.1080/01652176.2012.709952
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Veterinary Quarterly 2012, 1–6, iFirst
CASE REPORT Kiricephalus coarctatus in an Eastern Indigo Snake (Drymarchon couperi); endoscopic removal, identification, and phylogeny. A. Paige Brocka*, Alexander E. Gallaghera, Heather D. Stockdale Waldenb, Jennifer L. Owenc, Mark D. Dunbarc, Heather L. Wamsleyc, Amber B. Schoellera, April L. Childressa and James F.X. Wellehan Jra a Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA; bDepartment of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA; cDepartment of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
(Received 31 May 2012; final version received 4 July 2012)
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Keywords: Drymarchon couperi; Kiricephalus coarctatus; pentastomid; phylogenetics; pulmonoscopy
A wild adult male Eastern Indigo snake presented to the Zoological Medicine Service at the University of Florida Veterinary Hospital (UFVH) for the placement of a radio transmitter within his coelomic cavity as part of an ongoing study on habitat use. The surgery was performed and the snake recovered without complication. As part of the habitat-use study, the snake was tracked weekly and was sighted on several occasions. Two months after transmitter implantation, the snake was observed to have brown mucoid oral and nasal discharge. Five months after presentation, the snake was recaptured, noted to have a 455 g weight loss (24% of initial body weight) and returned to the UFVH for diagnostics. Upon presentation, the snake was bright, alert, and responsive. The body condition was considered poor. No nasal or oral discharge was noted. The remainder of the physical exam was within normal limits. Blood was collected from the tail vein and submitted for a blood smear analysis which revealed a heterophilia of 12.2 ! 103/mL (reference range, 1.68 " 1.67 ! 103/mL) and monocytosis of 6.6 ! 103/mL (reference range, 1.19 " 1.42 ! 103/mL) (International Species Information System (ISIS) 2003). Rare hemogregarines were also noted. A pulmonary wash was performed with 4 mL of sterile saline and a red rubber catheter. At the time of collection it was suspected that the sample was contaminated by the oral cavity. A second sample was collected as the snake was held vertically so that remaining fluid drained out of the lungs and trachea and through the mouth and nares. Cytology of the first sample revealed normalappearing ciliated, columnar, respiratory epithelial cells which were occasionally seen in moderately *Corresponding author. Email:
[email protected] ISSN 0165–2176 print/ISSN 1875–5941 online ! 2012 Taylor & Francis http://dx.doi.org/10.1080/01652176.2012.709952 http://www.tandfonline.com
sized clumps. Extracellular monomorphic bacilli and focal areas of abundant mineral were also seen. A few mature, non-degenerate heterophils, non-vacuolated macrophages, and small, well-differentiated lymphocytes were observed. A wet mount of the second sample revealed a dense mucoid background with minimal cellularity (Figure 1). A few egg shells were seen, most of which were cracked and empty. A low number of the eggs contained an organism, exhibiting varying stages of larval development. Hatched larvae with morphology consistent with the pentastomid Kiricephalus coarctatus were observed moving through the mucoid material. The organisms were approximately 120 mm long and had four legs with two claws at each end. A small ‘‘spear’’ or ‘‘boring apparatus’’ was noted at the apex of the larva’s head and the tail was stubby with a rounded end. Based on cytology, a patent infestation of pentastomids was suspected and exploration of the respiratory tract with manual removal of the adult pentastomids using endoscopy was elected. The indigo snake was sedated with 50 mg/kg BW of dexmedetomidine, 5 mg/kg BW of ketamine, and midazolam 0.1 mg/kg BW IM to allow placement of an endotracheal tube. It was then induced with 5% isoflurane in 100% oxygen (3 L/min) and maintained with 0.5–1.0% isoflurane. The snake was placed in right lateral recumbency and the caudal extent of the respiratory tract was identified by providing positive pressure ventilation. An incision site was selected 20 cm rostral to the caudal-most extent. A 2 cm longitudinal skin incision was made between the first and second row of scales dorsolateral to the ventral scutes. The subcutaneous tissues were bluntly dissected. The intercostal muscles were
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Figure 1. Pulmonary wash from an indigo snake containing larvated and unlarvated eggs, empty egg shells and free moving larvae (inset) consistent with multiple stages of Kiricephalus coarctatus. Bar ¼ 100 mm; Inset bar ¼ 50 mm.
penetrated with a Kelly hemostat and the adjacent ribs separated. A Weitlaner retractor was placed and the air sac was identified. A small rent was created in the air sac and a rigid scope1 introduced into the lung. Initial examination of the air sac revealed three pentastomids within the air sac cranial to the incision. The parasites were removed intact by grasping each parasite with an endoscopic grasping forceps placed through the instrument channel of the scope sheath. The scope, forceps, and parasites were then withdrawn through the incision. The rigid scope was removed and a flexible bronchoscope2 inserted to allow further evaluation of the respiratory tract. The scope was passed rostrally, where two additional pentastomids were identified. Both were attached to the air sac membrane with tissue covering the head of the parasite. Removal of these additional pentastomids was attempted using the above technique. However, the pentastomids were firmly attached and the forceps partially tore the body of the parasite. A second removal attempt was made by bluntly dissecting the tissue over the head with the forceps and then detaching the parasites for removal. See the online Supplementary Content for a video at http:// dx.doi.org/10.1080/01652176.2012.709952. The scope was then passed rostrally through the right lung and into the trachea. No abnormalities were noted. The scope was retracted to the incision site and passed to the caudal extent of the air sac where two additional pentastomids were identified and removed. After the final pentastomid extraction the scope was removed. We chose not to close the rent in the airsac based on a previous study (Stahl et al. 2008). The ribs and intercostal musculature were opposed with simple
interrupted sutures using 3–0 polydioxanone suture. The skin was closed using 3–0 polydioxanone suture in a horizontal mattress pattern. Anesthesia was partially reversed with 0.0045 mg/ kg BW of flumazenil and 0.5 mg/kg BW of atipamezole administered IM. Meloxicam (0.2 mg/kg BW) was administered IM intraoperatively for analgesia. Upon recovery, hydromorphone (0.2 mg/kg BW) was also administered IM for analgesia. The snake recovered from anesthesia and pulmonoscopy without complication. The indigo snake was returned to the field biologists three days after the pulmonoscopy procedure and released at the capture site within the next week. Field biologists continued to track the snake for about 2 months at which time the radio transmitter failed and the snake was lost to follow-up. K. coarctatus is a pentastomid known to use a number of snakes including Eastern Indigo Snakes as hosts (Riley and Self 1980; Foster et al. 2000). The life cycle requires infestation of a primary intermediate host (mammal, amphibian, and lizard) through ingestion of the infective eggs. Snakes serve as secondary intermediate hosts through consumption of infective nymphs in primary intermediate hosts, as well as definitive hosts through ophiophagy of a secondary intermediate host (Riley and Self 1980). Eastern Indigo Snakes are ophiophagous, allowing this species to act as secondary intermediate and definitive hosts. K. coarctatus is typically found in the lungs or body cavity of snakes acting as the definitive host (Detterline et al. 1984; Foster et al. 2000). K. coarctatus has been documented to cause airway obstruction, although this diagnosis may be complicated by the fact that parasites have been observed to leave dead or dying hosts by
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Figure 2. Adult male (right) and female (left) Kiricephalus coarctatus removed from an Eastern Indigo snake.
exiting through the mouth or nares (Montgomery et al. 2006). Identification of pentastomids is frequently based on morphologic characteristics. However, genetic characterization of many taxa has resulted in better phylogenetic resolution and identification of cryptic species (Almeida and Christoffersen 1999). There has been a lack of sequence-based phylogeny of Pentastomida. In this case report, we present the endoscopic treatment of a case of K. coarctatus infestation in an Eastern Indigo snake and the first sequence-based phylogenetic tree of pentastomids. Three adult pentastomids were submitted for morphologic identification. K. coarctatus infestation was diagnosed based on numerous morphological characteristics consistent with those previously described (Riley and Self 1980). The swollen, globular cephalothorax was observed in both male and female specimens and was flattened ventrally. The female also demonstrated the characteristic neck-line constriction that is not as evident in the male. Both male and female specimens had two pairs of hooks with the mouth located between the inner pair of hooks. The female was 84 mm in length and had 50 annuli, while the male was 30 mm in length with 48 annuli (Figure 2). The K. coarctatus were submitted for PCR, sequencing, and phylogenetic analysis. DNA was extracted from a K. coarctatus using a commercial
kit.3 PCR methods previously described for pentastomids were used, with primers designed from conserved regions of the pentastomid small subunit ribosomal RNA gene (SSU) (Brookins et al. 2009). The PCR products were resolved in 1% agarose gels. The bands were excised and purified using a gel extraction kit.4 Direct sequencing was performed5 and analyzed on automated DNA sequencers6 at the University of Florida Interdisciplinary Center for Biotechnology Research Sequencing Facilities, Gainesville, Florida. All products were sequenced in both directions. Primer sequences were edited out prior to further analysis. The PCR amplification of K. coarctatus produced a 383 nucleotide product when primer sequences were edited out. The sequence was submitted to GenBank under accession number JQ617282. Predicted homologous 376–383 nucleotide sequences of the 18S rRNA gene were aligned using MAFFT (Katoh and Toh 2008). Normplatnicka barrettae (GenBank accession number HM357505) and Bentheuphausia amblyops (DQ900734), non-pentastomid arthropods, were included. N. barrettae was used as an outgroup. Bayesian analyses of nucleotide alignments were performed using MrBayes 3.1 (Ronquist and Huelsenbeck 2003) on the CIPRES server (Miller et al. 2010) with gamma distributed rate variation and a proportion of invariant sites, and a general time reversible substitution model. Four chains were run
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Figure 3. Bayesian phylogenetic tree of predicted homologous 376–383 nucleotide sequences of the 18S rRNA gene of pentastomids. Genbank accession numbers follow the names. Bayesian posterior probabilities of branchings, as percentages, are in bold on the left and ML bootstrap values for branchings are given to the right. Pentastomid orders are marked with brackets. An arrow marks Kiricephalus coarctatus.
and statistical convergence was assessed by looking at the standard deviation of split frequencies as well as potential scale reduction factors of parameters. The first 10% of 2,000,000 iterations were discarded as a burn in. The Bayesian tree is shown (Figure 3). Stopping criteria for maximum likelihood (ML) bootstrapping were reached after 350 subsets. ML analyses of each alignment were performed using RAxML on the CIPRES server (Stamatakis et al. 2008) with gamma distributed rate variation and a proportion of invariant sites, and a general time reversible substitution model. Bootstrap analysis was used to test the strength of the tree topology (Felsenstein 1985). Numbers of bootstrap replicates were determined using previously described stopping criteria (Pattengale et al. 2010). Bootstrap values as percentages from ML analysis are shown on the Bayesian tree (Figure 3). K. coarctatus was found to form a well-supported clade with Armillifer armillatus and Porocephalus crotali using both Bayesian and ML methods. This case indicates that snakes which are infested as definitive hosts by K. coarctatus may present with intermittent respiratory signs. This infestation may have been responsible for the snake’s poor body
condition; however, this cannot be confirmed. Additionally, this case illustrates that a single negative tracheal or pulmonary wash does not exclude the possibility of a pentastomid infestation even in cases with patent infestations. Controlled data on pharmacologic treatment of pentastomid infestation is very limited. In one study, ivermectin was found to successfully treat Linguatula arctica adults in their definitive host, reindeer (Rangifer tarandus) (Haugerud et al. 1993). However, another study found that ivermectin therapy failed to eradicate L. arctica in reindeer (Nilssen et al. 2002). Even if anthelmintic treatment is successful, complications caused by dead parasites are a concern, and surgical removal may, therefore, be a better option. In humans, the nasopharyngeal pentastomiasis has been reported in at least two cases. However, most reported cases are the visceral form which occurs through infestation by ingestion of a primary intermediate host. Treatment of clinical visceral pentastomiasis in humans is usually based on surgical removal while treatment of nasopharyngeal pentastomiasis has been limited to local washes (Machado et al. 2006; Yao et al. 2008). Endoscopy through an airsac has been described for diagnostic purposes (Jekl and Knotek 2006) as well
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Veterinary Quarterly as a method of Porocephalus clavatus removal in boa constrictors (Foldenauer et al. 2008). In the boa constrictor case series, a cadaver boa from the same collection was used to determine the surgical approach and describe the respiratory anatomy prior to endoscopy of the infested boas; however, no such opportunity was available in the indigo snake case. Surgical approach was selected somewhat subjectively and based on the reports in other snake species, but resulted in successful endoscopic examination of the respiratory tract in this individual. This case shows that transcutaneous pulmonoscopy is also an effective means of removing adult K. coarctatus from indigo snakes. Based on the success in boas and the current case as well as the lack of demonstrated efficacy of systemic anthelmintic agents, transcutaneous pulmonoscopy may be the preferred first-line therapy for adult pentastomid infections in snakes. Identification of the pentastomid species was important as it allowed assessment of the zoonotic potential of this parasite. Armillifer armillatus and P. crotali have both been shown to cause significant disease in mammalian intermediate hosts (Lavarde and Fornes 1999; Brookins et al. 2009). The finding that K. coarctatus is in a well-supported clade with these species raises the level of concern about its zoonotic potential. The SSU phylogenetic analysis found that Reighardia, Raillietiella, and Hispania also form a well-supported clade. This contrasts with a previous analysis based on morphologic data, in which Reighardia was considered more closely related to Kiricephalus, Armillifer, and Porocephalus in a clade called Reighardiida that did not include Raillietiella (Almeida and Christoffersen 1999). Acknowledgments The authors would like to thank the Orianne Society for their support in the diagnosis and treatment of this case.
Notes 1. Storz rigid endoscope 4.0 mm ! 30 cm with 17.5 fr sheath, Karl Storz GmbH & Co. KG, Tuttlingen, Germany 2. Storz 4.9 mm ! 85 cm fiberscope, Karl Storz GmbH & Co. KG, Tuttlingen, Germany 3. DNEasy tissue kit, QIAGEN Inc., Valencia, CA 91255, USA 4. QIAquick gel extraction kit, QIAGEN Inc., Valencia, CA 91255, USA 5. Big-Dye Terminator Kit, Applied Biosystems, Foster City, CA 94404, USA 6. ABI 3130 automated DNA sequencer, Applied Biosystems, Foster City, CA 94404, USA
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