Cytobrushing of the oral mucosa as a possible tool for early detection of testudinid herpesvirus in Horsfield’s tortoises with nonspecific clinical signs

Forty-five Horsfield’s tortoises (Testudo horsfieldii; syn. Agrionemys horfieldii, Russian tortoise) belonging to different owners had decreased appetite and respiratory issues. Twenty-nine tortoises had epiphora, dyspnea, and white necrotic diphtheroid oral plaques (group G1). Ten of the remaining 16 tortoises had serious dehydration, appetite disorder, and depression (G2). The last 6 tortoises had only decreased appetite and moderate conjunctival discharge (G3). During the physical examination of all 45 tortoises, a cytologic sample and an oral swab for herpesvirus and Mycoplasma agassizii PCR testing were taken. In 20 of 29 specimens from G1, in 8 of 16 from G2, and 0 of 6 from G3, the cytologic exam revealed intranuclear acidophilic inclusion bodies, multinucleate cellular syncytia, and further abnormalities caused by herpesviral infection. Moreover, all 45 tested subjects were found to be positive for testudinid herpesvirus 1; 2 were positive for M. agassizii. This prospective study suggests that Horsfield’s tortoises with such signs would benefit from this screening procedure, given that it was effective in a significant proportion of infected and symptomatic animals, and no negative effects were seen.     

Mojave desert tortoise (Gopherus agassizii) thermal ecology and reproductive success along a rainfall cline

Desert resource environments (e.g. microclimates, food) are tied to limited, highly localized rainfall regimes which generate microgeographic variation in the life histories of inhabitants. Typically, enhanced growth rates, reproduction and survivorship are observed in response to increased resource availability in a variety of desert plants and short‐lived animals. We examined the thermal ecology and reproduction of US federally threatened Mojave desert tortoises (Gopherus agassizii), long‐lived and large‐bodied ectotherms, at opposite ends of a 250‐m elevation‐related rainfall cline within Ivanpah Valley in the eastern Mojave Desert, California, USA. Biophysical operative environments in both the upper‐elevation, “Cima,” and the lower‐elevation, “Pumphouse,” plots corresponded with daily and seasonal patterns of incident solar radiation. Cima received 22% more rainfall and contained greater perennial vegetative cover, which conferred 5°C‐cooler daytime shaded temperatures. In a monitored average rainfall year, Cima tortoises had longer potential activity periods by up to several hours and greater ephemeral forage. Enhanced resource availability in Cima was associated with larger‐bodied females producing larger eggs, while still producing the same number of eggs as Pumphouse females. However, reproductive success was lower in Cima because 90% of eggs were depredated versus 11% in Pumphouse, indicating that predatory interactions produced counter‐gradient variation in reproductive success across the rainfall cline. Land‐use impacts on deserts (e.g. solar energy generation) are increasing rapidly, and conservation strategies designed to protect and recover threatened desert inhabitants, such as desert tortoises, should incorporate these strong ecosystem‐level responses to regional resource variation in assessments of habitat for prospective development and mitigation efforts.     

Identification of a novel herpesvirus from a California desert tortoise (Gopherus agassizii)

Herpesviruses are significant pathogens of tortoises, causing upper respiratory tract disease and necrotizing stomatitis, with infections often associated with high mortality rates. Herpesvirus infection in a captive California desert tortoise (Gopherus agassizii) was detected by light microscopic observation of intranuclear inclusion bodies in various tissues followed by transmission electron microscopic observation of herpesvirus-like particles, and amplification of herpesvirus nucleic acid sequences using polymerase chain reaction. Using an indirect enzyme linked immunosorbent assay, anti-tortoise herpesvirus antibodies were detected one month after initial onset of clinical signs. This novel herpesvirus is distinct from the previously described tortoise herpesvirus (tortoise herpesvirus-1, THV-1) sharing 83% sequence identity of 60 amino acids of a portion of the DNA polymerase gene and 79% sequence identity across 120 amino acids of a portion of the ribonucleotide reductase gene. Similar to THV-1, this novel herpesvirus, tortoise herpesvirus-2 (THV-2), also clusters with the alphaherpesviruses.     

Population pharmacokinetics of a single intramuscular administration of tulathromycin in adult desert tortoises (Gopherus agassizii)

Tulathromycin, a long acting macrolide antibiotic, has demonstrated efficacy against respiratory pathogens including Mycoplasma bovis and M. hyopneumoniae. A pharmacokinetic study was performed to evaluate the clinical applicability of tulathromycin in desert tortoises following a single intramuscular dose of 5 mg/kg. A single blood sample was collected from 110 different desert tortoises at 0.25, 0.5, 1, 4, 8, 24, 48, 72, 120, and 240 h following drug administration. Plasma concentrations of the parent form of tulathromycin were measured using liquid chromatography/mass spectrometry. As each tortoise was only bled once, pharmacokinetic parameters were initially estimated using a naïve pooled data approach. Given the variability in the data, population‐based compartmental modeling was also performed. Using nonparametric population compartmental modeling, a two‐compartment model with first‐order absorption and elimination best fit the data. An observed C_max of 36.2 ± 29.7 μg/mL was detected at 0.25 h (observed T_max). The elimination half‐life (T_½el) was long (77.1 h) resulting in detectable plasma concentrations 240 h postadministration. This study represents a preliminary step in evaluating the utility of tulathromycin in chelonian species and demonstrates that population data modeling offers advantages for estimating pharmacokinetic parameters where sparse data sampling occurs and there is substantial variability in the data.     

Subconjunctival Dirofilaria repens infection in a dog resident in the UK

Dirofilaria repens infection was diagnosed in a 5‐year‐old female German shepherd crossbreed, originally from Romania but brought into the UK in February 2014. The dog presented with conjunctivitis in March 2014 and then again 2 months later with additional ocular and nasal mucopurulent discharge. Bacterial cultures from the nasolacrimal duct were negative for bacterial growth. The case was referred in August 2014 for ophthalmic examination, which revealed abnormalities in both eyes, especially the left. They included mild palpebral conjunctival hyperaemia and marked follicular conjunctivitis, as well as a dorsonasal bulbar conjunctival mass. Serum biochemistry was unremarkable and a conjunctival biopsy taken from the dorsonasal bulbar conjunctival mass revealed eosinophilic/lymphoplasmacytic conjunctivitis. At re‐examination, nematodes were found in the area of the previous biopsy site and in the ventral palpebral conjunctival fornix. Polymerase chain reaction and sequencing confirmed these to be D. repens. Treatment with 10% imidacloprid and 2·5% moxidectin (Advocate Spot‐On) was successful, and clinical signs resolved over a 6‐week period. This case report indicates that D. repens infection should be considered as a possible aetiological cause of ocular lesions in dogs in the UK, especially those with a history of foreign travel. Implications for establishment and spread of D. repens in the UK are discussed.     

Diseases of the respiratory tract of chelonians.

Diseases of the respiratory tract commonly occur in captive chelonians, and several diseases also have occurred in wild chelonians. Infectious causes include viruses, bacteria, fungi, and parasites. Herpesviruses have surfaced as important pathogens of the oral cavity and respiratory tract in Hermann's tortoise (Testudo hermanii), spur-thighed tortoise (Testudo graeca), and other tortoises in Europe and the United States. Herpesvirus-associated respiratory diseases also have been reported in the green turtle, Chelonia mydas, in mariculture in the Cayman Islands. Of diseases caused by bacteria, an upper respiratory tract disease caused by Mycoplasma sp has been reported in free-hanging and captive gopher tortoises in the southeastern United States and in desert tortoises in the Mojave Desert of the southwestern United States. Mycotic pulmonary disease is commonly reported in captive chelonians, especially in those maintained at suboptimal temperatures. An intranuclear coccidia has been seen in several species of captive tortoises in the United States, and, in one case, a severe proliferative pneumonia was associated with organisms in the lung. The most common noninfectious cause of respiratory disease in chelonians results from trauma to the carapace. Although pulmonary fibromas commonly occur in green turtles with fibropapillomatosis, for the most part, tumors of the respiratory tract are uncommon in chelonians.     

Comparison of intraosseous and peripheral venous fluid dynamics in the desert tortoise (Gopherus agassizii)

The efficacy of intraosseous catheterization has not been described previously in the desert tortoise (Gopherus agassizii). The goal of this study was to describe and compare the efficacy of four intraosseous catheter sites (humerus, femur, plastocarapacial junction [bridge], and gular region of the plastron) to jugular catheterization. Five adult tortoises were catheterized in each of the sites at least once. The distribution of a bolus injection of radiopharmaceutical (technetium-99m-diethylenetriaminepentaacidic acid [99mTc -DTPA]) was monitored via gamma camera over 2-min periods at five time intervals over 24 min. Compared to jugular catheterization, the humerus and femur sites provided the next best vascular access, with 84.4 and 61.8% of activity reaching the systemic circulation by 7 min, respectively. The bridge and gular catheter sites were less effective with only 41.9 and 40.8% systemic activity, respectively. Intraosseous catheters were no more technically difficult to place than jugular catheters and were less commonly dislodged, making them a viable option for vascular access in tortoises.     

Osseous metaplasia in the conjunctiva of a horse presenting with recurrent ulcerative keratitis

A 13‐year‐old Thoroughbred gelding presented with a 6 month history of recurrent conjunctivitis, dacryocystitis and corneal ulceration affecting the left eye (OS). Treatment had included multiple topical antibiotic and anti‐inflammatory preparations, nasolacrimal system flushing and an injection of methylprednisolone acetate (MPA) (Depomedrone) ¹ administered subconjunctivally into the left lower eyelid. No mechanical cause for the ulcerative keratitis could be visualised but digital palpation revealed a firm linear structure in the central palpebral conjunctiva of the lower eyelid. An excisional biopsy was performed and histopathological examination revealed the lesion to be a region of osseous metaplasia. Following excision of the lesion, the corneal ulcer resolved and the eye remained disease free over a 4 year follow‐up period. A causal link between the osseous metaplasia and the recurrent ulcerative keratitis was therefore suspected. This report describes the management of this case and discusses the pathogenesis of osseous metaplasia and the use of subconjunctival corticosteroids.     

A case of lipogranulomatous conjunctivitis in a horse

Objective

To describe a case of lipogranulomatous conjunctivitis in a horse.

Animal Studied

A client-owned 12-year-old Standardbred gelding presenting with chronic conjunctivitis and palpebral masses.

Methods

Complete ophthalmic examination, surgical excision, histopathology, and bacterial culture of biopsy samples were performed.

Results

Upper and lower eyelids of both eyes were affected, with multiple yellow-to-white nodules on the palpebral conjunctiva, adjacent to the eyelid margin. Nodule removal was achieved via partial tarsal plate excision. Histopathological examination revealed granulomatous inflammation and large droplets of presumed free lipid in the conjunctival lamina propria. The animal was diagnosed with lipogranulomatous conjunctivitis. No postoperative ocular discomfort was observed for months; however, 3 years later, new conjunctival nodules were noticed, requiring a second surgical procedure on three of the eyelids. Transient entropion in the left lower eyelid was observed 2 months after the second surgery, and no recurrence of conjunctival nodules was observed after 18 months of follow-up.

Conclusion

To our knowledge, this is the first report of lipogranulomatous conjunctivitis in horses.     

Evaluation of cytologic findings in feline conjunctivitis

Background

Cytologic examination of smears prepared from ocular swabs of conjunctiva from cats with conjunctivitis permits identification of the type of inflammation and possibly specific microorganisms. Results of studies of the diagnostic utility of cytology for detection of infectious causes of feline conjunctivitis have been inconsistent.

Objectives

The objectives of this study were to describe cytologic findings in cats with conjunctivitis and to compare those findings with results of PCR analysis for feline herpesvirus (FHV‐1), Chlamydophila felis (C felis), and Mycoplasma felis (M felis).

Methods

Conjunctival smears from 88 cats with conjunctivitis and 10 healthy control cats were stained with a Romanowsky stain and evaluated for the type of inflammation and evidence of an infectious agent. PCR analysis for FHV‐1, C felis, and M felis was performed.

Results

Infectious agents identified by PCR analysis were FHV‐1 in 9 cats (10%), C felis in 8 cats (9%), and M felis in 6 cats (7%). Inclusions interpreted as chlamydial inclusions were found in all cytologic smears from cats positive for C felis by PCR analysis and in 3 PCR‐negative cats. Inclusions interpreted as Mycoplasma organisms were found in 3 of 6 cats that were PCR‐positive for M felis and in 1 PCR‐negative cat. FHV‐1 inclusion bodies were not detected on cytologic examination.

Conclusions

Cytologic examination can be diagnostic for C felis infection when many typical inclusions are present. Cytologic examination was unreliable in diagnosing M felis infection, and viral inclusions of FHV‐1 were not found in specimens stained with Romanowsky stains.     

Morphologic and cytochemical characteristics of blood cells from the desert tortoise (Gopherus agassizii).

Morphologic and cytochemical staining characteristics of erythrocytes, leukocytes, and thrombocytes of the desert tortoise (Gopherus agassizii) were evaluated, using blood smears prepared from 23 healthy tortoises of Kern County, Calif. Special emphasis was placed on differentiating features of the various leukocytes and thrombocytes. A variety of cytochemical stains, including benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff, and toluidine blue were used. Heterophils had a characteristic, large, focal area of positive staining with chloroacetate esterase, alpha-naphthyl butyrate esterase, and acid phosphatase. Eosinophils stained diffusely positive with benzidine peroxidase, allowing differentiation of this leukocyte from heterophils. Thrombocytes stained focally positive with periodic acid-Schiff, allowing differentiation of these cells from lymphocytes, which stained uniformly negative. An intracytoplasmic body, commonly observed within erythrocytes, was considered ultrastructurally to represent a degenerate organelle.     

Characterization of experimental Mycoplasma gallisepticum infection in captive house finch flocks

The use of controlled, horizontal-transmission experiments provides detailed information on the spread of disease within fixed social groups, which informs our understanding of disease dynamics both in an empirical and theoretical context. For that reason, we characterized in 2002, horizontal transmission of Mycoplasma gallisepticum (MG) in two flocks of 11 wild-caught house finches housed in outdoor aviaries over a 6-mo period. All birds were initially free of MG by a polymerase chain reaction (PCR)-based test, rapid plate agglutination (RPA), and the scoring of physical signs. We inoculated one flock member bilaterally in the palpebral conjunctiva and reintroduced it into its cage. Index birds developed conjunctivitis within 3 to 5 days but died 13 and 20 days postinfection (PI) possibly because of very severe weather. The proportion of birds with physical signs increased gradually, reached 40% at 6 wk PI, and fluctuated around 40% until 21 wk PI. By the time our experiment ended at 24.5 wk PI, 28% of the birds still exhibited physical signs. Across both flocks, 80% of the birds developed unilateral or bilateral conjunctivitis, and several birds relapsed. The appearance of physical signs in new individuals occurred between 10 and 144 days PI (median 41 days PI). Physical signs lasted 1-172 days (median 42 days). Birds that became infected earlier during the experiment developed more severe conjunctivitis, and there was a tendency for birds that developed bilateral conjunctivitis to develop physical signs earlier. Most birds that developed physical signs of MG were also PCR- and RPA-positive, although we detected a single asymptomatic carrier and a single symptomatic false negative. No birds died as a result of secondary MG infection.     

Persistence of maternal antibodies against Mycoplasma agassizii in desert tortoise hatchlings.

OBJECTIVE: To investigate Mycoplasma agassizii-specific maternal antibodies in desert tortoise (Gopherus agassizii) hatchlings. SAMPLE POPULATION: Plasma from 43 captive-reared desert tortoise hatchlings. PROCEDURE: ELISA for M agassizii-specific antibodies was performed. Four hatchlings from 4 clutches of 3 M agassizii-seropositive females with chronic upper respiratory tract disease (URTD) were tested on the day of hatching (set 1), and 20 hatchlings from 4 clutches of 4 M agassizii-seropositive females with URTD and 19 hatchlings from 4 M agassizii-seronegative healthy females were tested at 4, 8, 12, and 29 months old (set 2). Immunoblot analysis was performed to determine immunoglobulin classes in yolk and plasma of hatchlings. To determine infection status of hatchlings, yolk, egg shell membranes (set 1), and nasal lavage fluid (sets 1 and 2) were examined for M agassizii by use of polymerase chain reaction. RESULTS: Yolk and hatchling plasma had significantly lower amounts of specific antibodies than did plasma from adult females. The IgG and IgM antibodies were transferred, but M agassizii-specific antibodies were of the IgG class. Hatchlings were not infected with mycoplasmas. Offspring of sick females had significantly higher specific antibody titers than did offspring of healthy females. Titers were still significantly different in 1-year-old hatchlings. CONCLUSIONS: Desert tortoise females transfer specific IgG and IgM antibodies to their offspring that are still detectable after 1 year. CLINICAL RELEVANCE: Infection with M agassizii may be misdiagnosed in hatchlings with persistent maternal antibodies. Passively acquired antibodies may have a role in pathogenesis of mycoplasma-induced respiratory tract disease and other diseases.     

Treatment of Advanced Squamous Cell Carcinomas Involving the Equine Cornea

Corneal squamous cell carcinomas with surface areas of more than 2.0 cm were diagnosed in 26 eyes of 25 horses. The depth of the lesion into the corneal stroma and involvement of the ocular adnexa were important determinants for surgical treatment. Corneal tumors that extended into adjacent palpebral conjunctiva, eyelid, or orbit were not amenable to complete excision, while those that involved only cornea and bulbar conjunctiva were treated surgically by keratectomy and beta radiation. Initial treatment was keratectomy followed by beta radiation on 24 eyes and enucleation of two eyes. Twenty keratectomy patients remained tumor free after long-term follow-up. One was lost to follow-up and three recurred. The recurrences were retreated with subsequent cure (one horse), enucleation (one horse), or euthanasia (one horse).     

Clarithromycin pharmacokinetics in the desert tortoise (Gopherus agassizii).

Clarithromycin is a new, safe orally administered macrolide antibiotic active against Mycoplasma sp. in humans. Single-dose and multidose pharmacokinetic parameters were determined for clarithromycin in wild-caught desert tortoises (Gopherus agassizii) seropositive for M. agassizii. Clarithromycin blood levels were measured in three tortoises for up to 72 hr after a single oral dose of 7.5 mg/kg. In a second group of six tortoises, levels were measured after a dose of 15 mg/kg. Noncompartmental iterative two-stage Bayesian and nonparametric expectation maximization pharmacokinetic parameters were determined for each animal assuming first order rate constants. At 15 mg/kg, the maximum concentration was 1.37 microg/ml, the time to maximum concentration was 8.0 hr, and a plasma half-life of 11.69 hr was derived from the latter method. The absorption constant was 0.08/hr, the absorption half-life was 8.47 hr, and the weight-normalized volume of distribution was 5.30 L/kg. Predictions derived by the latter method suggested a dosage of 15 mg/kg p.o. every 24 hr to achieve maximal blood levels of > or =1 microg/ml for multiple dosing. However, results from a preliminary multidose study with three tortoises indicate that the drug is accumulated; therefore, the predicted dose may be closer to 15 mg/kg p.o. every 2-3 days to maintain blood levels of 2-7.5 microg/ml. (For n = 3, 2-point linear regression median estimates for the apparent elimination rate constant (K) and half-life are 0.0227/hr and 30.52 hr, respectively.) This multidose accumulation reflects a slower apparent elimination than that predicted in the eight single-dose tortoises (i.e., K = 0.0593/hr, t1/2 = 11.69 hr). This study highlights a potential pitfall of depending solely on single-dose studies and the potential value of oral administration in reptiles.     

Feline eosinophilic conjunctivitis

Objective To review 12 cases of histologically confirmed feline eosinophilic conjunctivitis, their clinical, cytologic, histologic and electronmicroscopic findings, results on PCR for FeHV‐1, treatment and outcome. Animals studied Twelve naturally occurring cases presented during a period of 26 months. Procedures Thorough ophthalmologic examination, conjunctival scrapings performed with the cytobrush method; histologic samples from the palpebral conjunctiva; PCR for FeHV‐1 on Schirmer Tear Test (STT) strips; saliva and nasal swabs, and retrospective evaluation of all results. Results The breed most commonly affected was the Domestic Shorthair (n = 8), followed by Persians (n = 2), Somali (n = 1) and Siamese (n = 1). Age at presentation was 1–15 years with a mean age of 7.2 years. Nine cats were castrated males; three cats were females: two of them were spayed. Unilateral (n = 7) or bilateral (n = 5) involvement with depigmentation and erosions of lid margin, blepharospasm, swelling and redness of conjunctiva and third eyelid were the most common clinical findings. Frequency of eosinophils in cytologic samples was more than 10% in every patient. PCR for FeHV‐1 on STT was negative in all cases. Histologically, eosinophils, lymphocytes, plasma cells, mast cells and macrophages were involved. On electronmicroscopy, viral particles were not detected. Ten cases needed long‐term anti‐inflammatory treatment. Conclusions The 12 reviewed cases suggest that feline eosinophilic conjunctivitis is a chronic inflammatory uni‐ or bilateral disease of the adult cat. Typically the lid margin was also involved, and was thickened, depigmented and erosive. Cytological examination of conjunctival scrapings was a valuable tool for detecting eosinophilic conjunctivitis. The cytological findings correlated well with the histopathological findings in our patients. Topical or systemic anti‐inflammatory drugs resolved the clinical symptoms in our cases within a short period of time. Neither electronmicroscopy nor PCR were able to detect involvement of FHV1 in the represented cases. The etiopathogenic role of FeHV‐1 remains undetermined.     

Comparison of cytologic and histologic evaluations of the conjunctiva in the normal equine eye

OBJECTIVE: To describe the cells observed in conjunctival brush cytology (CBC) from normal horses and compare these findings with conjunctival structural histology so as to understand which cells are recovered from CBC. METHODS: This study was divided into three parts. (1) Conjunctival brush smears were collected from 20 healthy horses on both eyes and a differential count on 300 cells was carried out on May Grünwald-Giemsa (MGG) smears. (2) A similar protocol was used for whole eyes from five horses obtained rapidly after death from a slaughterhouse. The eyes were then assessed for conjunctival histology. (3) Cytobrush smears were collected from five healthy horses. Smears were examined after MGG or periodic acid Schiff (PAS) staining. RESULTS: The differential cell count showed a majority of deep and intermediate epithelial cells with very few superficial and goblet cells in both eyes. A stratified columnar to cuboidal epithelium was observed on nearly the whole surface of the conjunctiva. A stratified squamous type was observed at the palpebral and bulbar edges. Areas with highest mucus cell indices were found from the nasal to the temporal edge of the equine inferior conjunctiva in the upper palpebral segment near the fornix and in a part of the nasal fornix. In MGG smears no mucus cells were identified; however, they were numerous in PAS smears (22.6% +/- 11) and were mostly cylindrical cells (42.5% +/- 14.4 PAS positive). CONCLUSIONS: Cytobrush smears in the healthy horse are characterized by a majority of polyhedral and cylindrical cells and a few squamous cells. The cylindrical cells may be mucous cells and probably originate from the main stratified columnar to cuboidal epithelium.     

ULTRASONOGRAPHY OF THE CALIFORNIA DESERT TORTOISE (Xerobates agassizi): ANATOMY AND APPLICATION

Ultrasonographic examinations were performed on healthy California desert tortoises to evaluate the utility of the anatomic acoustic windows available and to gain information about the normal echoanatomy of the tortoise. A major objective was to evaluate the potential clinical value of this diagnostic tool in these species. The soft tissue areas of the integument were used as acoustic windows. Three imaging approaches were described as mediastinal, axillary, and inguinal openings. The mediastinal and axillary approaches offered good visualization of the heart, liver, and gallbladder. The inguinal window allowed imaging of the intestines, urinary/accessory bladders, kidneys, and gonads. The pancreas and spleen could not be visualized. A tortoise with pericardial effusion and a tortoise with hepatic lipidosis are presented to illustrate clinical applications of ultrasound in diagnosing diseases of the tortoise.