The association of bacterial agents and flagellated protozoans in the etiopathogenesis of subspectacular abscesses in snakes.

OBJECTIVES: To describe the etiology, clinical pathology, and treatment of subspectacular abscesses (SAs) in 16 snakes. ANIMALS STUDIED: Sixteen snakes from private captive collections that were presented at a veterinary teaching hospital and diagnosed with SAs. PROCEDURES: Exudate was collected from SAs via percutaneous aspiration or by performing a partial spectaculectomy and submitted to direct light microscopic examination and/or microbiological examination. RESULTS: Bacterial isolates were cultured from exudate samples in 12 out of 16 snakes and comprised Pseudomonas aeruginosa isolates that showed identical antimicrobial susceptibility patterns in seven cases. Direct light microscopic examination demonstrated the presence of flagellated protozoa in the exudate in 10 out of 16 snakes and in all of these snakes, concurrent oropharyngeal flagellated protozoan infection could be demonstrated. Combined flagellated protozoan and bacterial SA infection was demonstrated in six snakes. Comparison of small subunit ribosomal RNA gene sequences of flagellated protozoans that were detected in the exudate of SAs from four snakes revealed they represented the parabasalians Monocercomonas colubrorum and Hypotrichomonas acosta in one and three cases, respectively. In all snakes, successful treatment of the SAs was achieved following surgical debridement of the subspectacular space combined with antimicrobial treatment. CONCLUSION: Bacteria as well as flagellates are considered to reach the subspectacular space through ascending infection from the oral cavity via the lacrimal duct. Although opportunistic bacterial infection presumably constitutes the predominant cause of SAs in snakes, the role of flagellate infection in the pathogenesis of this disorder should be further elucidated.

PROTEIN ELECTROPHORESIS OF PLASMA SAMPLES FROM BOA CONSTRICTORS WITH AND WITHOUT REPTARENAVIRUS INFECTION.

Reptarenaviruses infect a variety of boid and pythonid snake species worldwide and have been shown to be the cause of inclusion body disease (IBD). Little is known about the correlations between virus infection and clinical disease, as well as the effects of viral infection on the immune system and the blood protein fractions. The goal of this study was to examine the differences in the plasma protein fractions in reptarenavirus reverse transcription polymerase chain reaction (RT-PCR)-negative and -positive tested snakes with and without clinical signs of disease. Blood from a total of 111 boa constrictors (Boa constrictor) was evaluated. Reverse transcription PCRs and H&E staining for inclusion bodies were carried out on each sample for the detection of reptarenavirus, and the plasma protein fractions were evaluated by capillary zone electrophoresis (CZE). Thirty four of the 111 evaluated snakes were positive by RT-PCR and 19 of the 34 showed clinical signs of disease. In comparison with IBD-negative healthy boa constrictors, the positive snakes with clinical signs had significantly lower albumin levels (P = 0.0052), lower A: G ratios (P = 0.0037), and lower α-globulin levels (P = 0.0073), while their γ-globulin levels were significantly higher (P = 0.0004). In the same comparison, clinically healthy arenavirus-positive boas showed only significantly lower α-globulin (P = 0.0124) and higher γ-globulin levels (P = 0.0394). The results of the present study indicate that reptarenavirus infection may influence plasma protein fractions in boa constrictors.

Isolation of Burkholderia pseudomallei from a Pet Green Iguana, Belgium.

We isolated Burkholderia pseudomallei, the causative agent of melioidosis, from liver granulomas of a pet green iguana (Iguana iguana) in Belgium. This case highlights a risk for imported green iguanas acting as a reservoir for introduction of this high-threat, zoonotic pathogen into nonendemic regions.

Co-occurrence of Mycoplasma Species and Pigeon Herpesvirus-1 Infection in Racing Pigeons ( Columba livia).

Oropharyngeal swab samples were collected from 438 live racing pigeons ( Columba livia), with and without signs of respiratory disease, that were housed in 220 lofts in 3 provinces in the western part of the Netherlands. Polymerase chain reaction (PCR) was used to identify Mycoplasma species and pigeon herpesvirus-1 (PHV-1) from the samples. In 8.6% of the pigeon lofts tested, signs of respiratory disease were present in pigeons at sampling, and in 30.9% of the sampled pigeon lofts, respiratory signs were observed in pigeons during the 6-month period immediately before sampling. A total of 39.8% of tested pigeons (54.5% of tested lofts) were positive for Mycoplasma species, and 30.6% of tested pigeons (48.6% of tested lofts) were positive for PHV-1. In 15.8% of the tested pigeons (26.8% of tested pigeon lofts), coinfection by Mycoplasma species and PHV-1 was identified. The number of pigeon lofts having pigeons coinfected by Mycoplasma species and PHV-1 was higher than that where only one of the infections was identified. Neither the presence of Mycoplasma species, PHV-1, nor the co-occurrence of both infections was significantly associated with signs of respiratory disease.

Marek's disease virus associated ocular lymphoma in Roulroul partridges (Rollulus rouloul).

Two 1-year old Roulroul partridges (Rollulus rouloul), one male and one female, were presented because of eye problems and anorexia. Twenty of the 30 Roulroul partridges in the owner's collection had already died. The affected birds stopped eating, became thinner, and eventually died. Antibiotic treatment, which started because of the suspicion of a septicaemic process, was unsuccessful. At clinical examination of the two partridges it was found that in both birds, one eye ball was filled with a whitish yellow amorphous material and the other eye ball of the female showed a distinct corneal opacity. Both presented birds were euthanized. Necropsy revealed no significant abnormalities in addition to the eye lesions. Histology and immunohistochemistry of the female's eye revealed an infiltrate of T-lymphocytes corresponding to ocular lymphoma. Herpesvirus genus-specific PCR, followed by Sanger sequencing confirmed the presumptive diagnosis of Marek's disease in both birds. To our knowledge, this is the first confirmed case of infection with Gallid Herpesvirus 2 (Marek's disease virus) in partridges and the first case in this specific species.

Intervertebral disk disease in 3 striped skunks (Mephitis mephitis).

OBJECTIVE: To describe diagnostic findings, surgical technique, and outcome in 3 striped skunks (Mephitis mephitis) with a history of paraparesis. STUDY DESIGN: Case series. ANIMALS: Skunks (n = 3) with paraparesis. METHODS: Neurologic examination revealed upper motor neuron disease (T2-L2) in 2 skunks and lower motor neuron disease (L3-S3) in 1 skunk. Diagnostic imaging included radiography, myelography, CT, and MRI and confirmed intervertebral disk herniation (IVDH) in each skunk. Because initial treatment with pain medication and cage rest did not result in lasting improvement, spinal surgery was performed. RESULTS: Hemilaminectomy (2 skunks) and dorsal laminectomy (1 skunk) was performed with removal of extruded disk material. The skunks improved after surgery but all had minor residual neurologic deficits when examined at various times postoperatively. CONCLUSION: Thoracolumbar intervertebral disk herniation occurs in skunks, and must be included in the differential diagnosis of paraparesis.

Mycoplasma columbinum Isolated From a Racing Pigeon ( Columba livia ) With Arthritis.

A juvenile racing pigeon ( Columba livia ) was presented with drooping of the wing and inability to fly. On physical examination, the right shoulder joint was swollen. The pigeon was euthanatized and submitted for necropsy. An excessive amount of fibrin was present in the canalis triosseus with severe arthritis of the affected shoulder joint. A pure growth of Mycoplasma-like colonies was obtained on microbiological culture of the shoulder joint. A 16S ribosomal RNA gene-specific polymerase chain reaction assay was performed on the isolate and revealed 100% similarity with Mycoplasma columbinum . Although infectious arthritis in homing pigeons is primarily associated with paratyphoid and Streptococcus gallolyticus infection, clinical practitioners should consider the potential role of Mycoplasma columbinum in arthritis in pigeons.

Anatomy, Physiology, and Disorders of the Spectacle, Subspectacular Space, and Its Lacrimal Drainage System in Squamates.

Various squamate species have completely fused eyelids that make up a transparent spectacle. The spectacle is a continuation of the integument that is renewed with each shedding cycle and creates a narrow subspectacular or corneospectacular space that is filled with lacrimal fluid. The latter is considered as the analogue of the conjunctival sac in other vertebrates. Almost all reptiles that have a spectacle lack a nictitating membrane, bursalis muscle, and lacrimal glands. The lacrimal fluid in the subspectacular space is secreted by the Harderian gland. The features of the spectacle and its lacrimal drainage system are an illustration of the enormous variation of the morphological adaptations that are seen in reptiles and one of the most distinguishable traits of snakes and most gecko species. Whereas ocular disease in squamates with a spectacle is infrequently seen in practice, disorders of the spectacle and the subspectacular space are commonly encountered. In order to apply an adequate diagnostic and therapeutic approach for these conditions, a sound knowledge and understanding of the anatomical and physiological peculiarities of the spectacle, subspectacular space, and lacrimal drainage system are fundamental.

Histological Variants of Squamous and Basal Cell Carcinoma in Squamates and Chelonians: A Comprehensive Classification.

In the present study, the histological characteristics of squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) obtained from 22 squamate and 13 chelonian species were retrospectively evaluated. While the examined tissues were originally diagnosed as 28 SCCs and 7 BCCs based on histological evaluation by a specialty diagnostic service, eight SCCs could be re-classified as BCCs and three SCCs proved to be non-neoplastic lesions. In addition, all SCCs and BCCs were classified into distinct histological variants. The SCCs could be categorized as one SCC in situ, three moderately differentiated SCCs, seven well-differentiated SCCs, and six keratoacanthomas. BCCs were classified as five solid BCCs, four infiltrating BCCs, five keratotic BCCs, and one basosquamous cell carcinoma. In addition, the present study reports the occurrence of BCCs in seven reptile species for the first time. In contrast to what has been documented in humans, IHC staining with the commercially available epithelial membrane antigen and epithelial antigen clone Ber-EP4 does not allow differentiation of SCCs from BCCs in reptiles, while cyclooxygenase-2 and E-cadherin staining seem to have discriminating potential. Although the gross pathological features of the examined SCCs and BCCs were highly similar, each tumor could be unequivocally assigned to a distinct histological variant according to the observed histological characteristics. Based on the results of this study, a histopathological classification for SCCs and BCCs is proposed, allowing accurate identification and differentiation of SCCs and BCCs and their histological variants in the examined reptile species. Presumably, BCCs are severely underdiagnosed in squamates and chelonians.

Gross, Histologic and Immunohistochemical Characteristics of Keratoacanthomas in Lizards.

The present study describes the clinical behavior as well as the histopathologic and immunohistochemical characteristics of keratoacanthomas (Kas) in three different saurian species. While Kas presented as two dermal lesions in a bearded dragon (Pogona vitticeps), multicentric Kas were observed in three panther chameleons (Furcifer pardalis) and a veiled chameleon (Chamaeleo calyptratus). Macroscopically, Kas presented as dome-shaped skin tumors with a centralized keratinous pearl and a diameter ranging from 0.1-1.5 cm. In all lizards, Kas were predominantly located at the dorsolateral body wall, and KA of the eyelid was additionally observed in three out of four chameleons. Histologically, KAs presented as relatively well-defined, circumscribed epidermal proliferations that consisted of a crateriform lesion containing a central keratinous pearl with minimally infiltrating borders. In all KAs, a consistent immunohistochemical pattern was observed, with the expression of cyclooxygenase-2, E-cadherin, and pan-cytokeratin. A follow-up period of one to two years was established in all lizards. While no recurrence was observed in the panther chameleons, recurrence of a single keratoacanthoma was observed in the bearded dragon after one year, and in the veiled chameleon, multicentric keratoacanthomas reappeared during a follow-up period of two years. We describe KA as a previously unrecognized neoplastic entity in lizards that constitutes a low-grade, non-invasive but rapidly growing skin tumor that may show a multicentric appearance, especially in chameleons. As previously postulated for dermal squamous cell carcinomas (SCC), artificial ultraviolet lighting may play an important role in the oncogenesis of KAs in lizards. Although dermal SCCs in lizards show similar predilection sites and gross pathologic features, our results suggest that KA should be considered as a histologic variant of SCC that represents a rather benign squamous proliferation in comparison to conventional SCCs. Early diagnosis of KA and reliable discrimination from SCCs are essential for the prognosis of this neoplastic entity in lizards.

Infection of juvenile falcons (Falco spp.) with intestinal Lawsonia intracellularis.

Intestinal infection of many host species with Lawsonia intracellularis are widely reported. Analyses of infections among carnivorous falcons have not previously been reported. Fifty juvenile captive falcons (Falco spp.) with or without Lawsonia infection were investigated in the United Arab Emirates, including clinical laboratory methods. Fresh intestinal biopsy samples were analysed by microbiological techniques for Lawsonia and other bacteria and by standard parasitological and pathological methods. Lawsonia intracellularis infection was diagnosed by microbiological examination and qPCR in 10 of 50 juvenile falcons at case examination. Seven of these 10 falcons were of normal clinical appearance, and the other three had other contributing factors to ill-thrift. A range of other conditions were noted in 40 case control falcons. This first report of Lawsonia infection in falcons suggests that the agent may have a limited contribution to clinical disease in these birds, including ill-thrift syndromes. This lack of clinical disease association mimics that noted among Lawsonia infections recorded in other avian families.

Establishment of a Real-Time PCR Assay for the Detection of Devriesea agamarum in Lizards.

(1) Background: Devriesea (D.) agamarum is a potential cause of dermatitis and cheilitis in lizards. The aim of this study was to establish a real-time PCR assay for the detection of D. agamarum. (2) Methods: Primers and probe were selected targeting the 16S rRNA gene, using sequences of 16S rRNA genes of D. agamarum as well as of other bacterial species derived from GenBank. The PCR assay was tested with 14 positive controls of different D. agamarum cultures as well as with 34 negative controls of various non-D. agamarum bacterial cultures. Additionally, samples of 38 lizards, mostly Uromastyx spp. and Pogona spp., submitted to a commercial veterinary laboratory were tested for the presence of D. agamarum using the established protocol. (3) Results: Concentrations of as low as 2 × 104 colonies per mL were detectable using dilutions of bacterial cell culture (corresponding to approximately 200 CFU per PCR). The assay resulted in an intraassay percent of coefficient of variation (CV) of 1.31% and an interassay CV of 1.80%. (4) Conclusions: The presented assay is able to detect D. agamarum in clinical samples, decreasing laboratory turn-around time in comparison to conventional culture-based detection methods.

The Use of Prefemoral Endoscope-Assisted Surgery and Transplastron Coeliotomy in Chelonian Reproductive Disorders.

Throughout the last decades, the increased popularity of the keeping of reptiles has led to a better understanding of the captive needs of a wide variety of species. Although this is reflected by the successful captive reproduction in many of those species, reproductive disorders such as preovulatory follicular stasis, postovulatory dystocia, secondary yolk coelomitis, and prolapse of the oviduct and male copulatory organ are commonly encountered in veterinary practice. In comparison to squamates, chelonians with postovulatory dystocia seem to be more responsive to oxytocin treatment, even in cases of chronic dystocia. There are various conditions, however, that necessitate the use of surgical procedures for the treatment of dystocia and other reproductive disorders in chelonians. Although restrictions may be encountered, the endoscope-assisted prefemoral approach is the least invasive and thus preferred technique instead of the ventral transplastron coeliotomy. The present report describes the diagnostic and surgical approach applied in seven cases of female chelonians with reproductive disorders. The therapeutic efficacy largely relied on the choice of minimally invasive endoscope-assisted surgery versus transplastron coeliotomy that was primarily dictated by the involved species, etiology, and associated pathology.

Cheilitis Associated with a Novel Herpesvirus in Two Panther Chameleons (Furcifer pardalis).

Intracellular epidermal inclusions were detected within histological sections of skin biopsies from two panther chameleons (Furcifer pardalis) with chronic cheilitis. Transmission electron microscopy (TEM) confirmed the abundant presence of icosahedral intracytoplasmic and intranuclear viral particles in infected keratinocytes, with an average diameter of 120-125 nm, consistent with herpesviruses (HVs). TEM also revealed the presence of virions in intercellular spaces and keratinocyte nuclei and features suggestive of capsid assembly, nuclear egress with primary envelopment and anterograde transport leading to virion assembly and release. Polymerase chain reaction (PCR) primers targeting a conserved region of herpesvirus DNA-dependent DNA polymerase were used to amplify and sequence a product from a nested HV PCR performed on skin biopsies of both chameleons. Comparative sequence analysis indicates that the virus detected in both chameleons was a novel member of the Alphaherpesvirinae, which we refer to as chamaeleonid herpesvirus 1 (chamHV 1). Based on the identical findings in both chameleons, we consider chamHV 1 to be a candidate aetiological agent of cheilitis in panther chameleons. This is the first report of skin lesions in a chameleon species associated with HV infection.

Cutaneous hyalohyphomycosis in a girdled lizard (Cordylus giganteus) caused by the Chrysosporium anamorph of Nannizziopsis vriesii and successful treatment with voriconazole.

The Chrysosporium anamorph of Nannizziopsis vriesii was associated with dermatomycosis and high mortality in a group of captive giant girdled lizards (Cordylus giganteus). Treatment of one of the infected girdled lizards with voriconazole, which was selected on the basis of in vitro sensitivity testing of the isolate, resulted in resolution of lesions and negative fungal cultures from the skin. Three hours after oral administration of 10 mg/kg, the plasma level of voriconazole exceeded the 0.25-µg/mL minimal inhibitory concentration tenfold. In conclusion, administration of voriconazole at 10 mg/kg of body weight once daily for 10 weeks resulted in clinical cure and was well tolerated. A longer follow-up time and larger studies will be necessary to determine the long-term efficacy and safety of this treatment in giant girdled lizards.

Prevalence of inclusion body disease and associated comorbidity in captive collections of boid and pythonid snakes in Belgium.

Inclusion body disease (IBD) is caused by reptarenaviruses and constitutes one of the most notorious viral diseases in snakes. Although central nervous system disease and various other clinical signs have been attributed to IBD in boid and pythonid snakes, studies that unambiguously reveal the clinical course of natural IBD and reptarenavirus infection are scarce. In the present study, the prevalence of IBD and reptarenaviruses in captive snake collections and the correlation of IBD and reptarenavirus infection with the clinical status of the sampled snakes were investigated. In three IBD positive collections, long-term follow-up during a three- to seven-year period was performed. A total of 292 snakes (178 boas and 114 pythons) from 40 collections in Belgium were sampled. In each snake, blood and buffy coat smears were evaluated for the presence of IBD inclusion bodies (IB) and whole blood was tested for reptarenavirus RNA by RT-PCR. Of all tested snakes, 16.5% (48/292) were positive for IBD of which all were boa constrictors (34.0%; 48/141) and 17.1% (50/292) were reptarenavirus RT-PCR positive. The presence of IB could not be demonstrated in any of the tested pythons, while 5.3% (6/114) were reptarenavirus positive. In contrast to pythons, the presence of IB in peripheral blood cells in boa constrictors is strongly correlated with reptarenavirus detection by RT-PCR (P<0.0001). Although boa constrictors often show persistent subclinical infection, long-term follow-up indicated that a considerable number (22.2%; 6/27) of IBD/reptarenavirus positive boas eventually develop IBD associated comorbidities.

Devriesea agamarum causes dermatitis in bearded dragons (Pogona vitticeps).

Devriesea agamarum is frequently isolated from dermatitis in lizards, notably from cheilitis in spiny tailed lizards (genus Uromastyx). It was the aim of the present study to assess the role of this bacterium as a causative agent of dermatitis by fulfilling Koch's postulates. First, its association with diseased lizards was demonstrated. The bacterium was isolated from several, mainly desert dwelling squamate species showing symptoms of dermatitis and/or septicaemia. The affected lizards mainly belonged to the family of the Agamidae (genera Pogona, Uromastyx, Agama) and in one case to the Iguanidae (genus Crotaphytus). Secondly, the occurrence of D. agamarum in 66 clinically healthy bearded dragons, 21 clinically healthy Uromastyx species and 40 squamate eggshells was studied. The bacterium was isolated from the oral cavity of 10 bearded dragons but from none of the healthy Uromastyx species. Hence D. agamarum was found to be part of the oral microbiota in Pogona vitticeps. Finally, bearded dragons (P. vitticeps) were experimentally inoculated with D. agamarum by direct application of a bacterial suspension on intact and abraded skin. At the scarified skin of all inoculated lizards, dermatitis was induced from which D. agamarum was re-isolated. In conclusion, D. agamarum is a facultative pathogenic bacterium, able to cause dermatitis in agamid lizards when the integrity of the skin is breached.

Treatment of otitis externa associated with Corynebacterium kroppenstedtii in a peach-faced lovebird (Agapornis roseicollis) with an acetic and boric acid commercial solution.

A 5-year-old lovebird (Agapornis roseicollis) was presented with scaly crusts around both external ear openings and exudate present around the left ear. The bird had been treated with ivermectin and enrofloxacin without success. A pure culture of Corynebacterium kroppenstedtii was isolated from both ears. After susceptibility testing, a treatment of an acetic and boric acid solution administered topically 3 times daily was prescribed. The scaly appearance disappeared after 14 days of treatment and C kroppenstedtii could not be reisolated.

Devriesea agamarum associated cheilitis in a North African spiny-tailed lizard (Uromastyx acanthinura) in Spain.

An 11-year-old, male North African spiny-tailed lizard (Uromastyx acanthinura) was presented with hyperkeratotic cheilitis. Based on clinical examination, histology and microbiological testing, Devriesea agamarum was identified as the causative agent of the dermal disorder. Moreover, an abundant culture of D. agamarum was obtained following sampling of the oral cavity. Cheilitis was present for three years in the spiny-tailed lizard and during the first year of that period the lizard was housed together with an ocellated lizard (Timon lepidus), an Algerian orange-tailed skink (Eumeces algeriensis) and a female U. acanthinura. The latter lizard showed signs of chronic dermatitis and had deceased approximately 2.5 years prior to initial presentation of the male dab lizard because of renal failure. The other cohabiting lizards showed no dermal lesions and D. agamarum could not be demonstrated following dermal, cloacal and oral sampling. Recurrence of the bacterial skin infection was observed following a first antimicrobial treatment. This was considered to result from failure to eliminate the bacterium from the treated dab lizard or re-infection from the environment or asymptomatic carriers. A second treatment, including disinfection of the enclosure with house-hold bleach 0.1%, weekly mechanical debridement of the crusty lesion and treatment with ceftazidime at 20 mg/kg intramuscularly every 72 hours for 57 days resulted in resolution of the skin lesions, elimination of D. agamarum from the oral cavity based on repetitive microbiological sampling and no recurrence of the lesions during a 31 month follow up period. The present case is the first report of devrieseasis in Spain and highlights the importance of a multidirectional diagnostic and therapeutic approach towards controlling devrieseasis in captive lizard collections. Several disease aspects such as persistency are discussed in the light of the contemporary available literature.