FDG PET/CT IMAGING IN CANINE CANCER PATIENTS

2‐Deoxy‐2‐[¹⁸F]fluoro‐d ‐glucose positron emission tomography/computed tomography (FDG PET/CT) is becoming increasingly available as an imaging modality in veterinary medicine. The purpose of this study was to report semiquantitative standard uptake values (SUV) of malignant and nonmalignant tissues and organs in canine cancer patients. FDG PET/CT was performed in 14 dogs including, nine mesenchymal tumors, four carcinomas, and one incompletely excised mast cell tumor. A generally higher FDG uptake was observed in carcinomas relative to sarcomas. Maximum SUV of carcinomas ranged from 7.6 to 27.0, and for sarcomas from 2.0 to 10.6. The FDG SUV of several organs and tissues, including regional brain uptake is reported, to serve as a reference for future FDG PET studies in canine cancer patients. Several potential pitfalls have been recognized in interpretation of FDG PET images of human patients, a number of these were also observed in this study.     

EFFECTS OF ANESTHETIC PROTOCOL ON NORMAL CANINE BRAIN UPTAKE OF 18F‐FDG ASSESSED BY PET/CT

The purpose of this study was to assess the effects of four anesthetic protocols on normal canine brain uptake of 2‐deoxy‐2‐[¹⁸F]fluoro‐d ‐glucose (FDG) using positron emission tomography/computed tomography (PET/CT). Five clinically normal beagle dogs were anesthetized with (1) propofol/isoflurane, (2) medetomidine/pentobarbital, (3) xylazine/ketamine, and (4) medetomidine/tiletamine–zolazepam in a randomized cross‐over design. The standard uptake value (SUV) of FDG was obtained in the frontal, parietal, temporal and occipital lobes, cerebellum, brainstem and whole brain, and compared within and between anesthetic protocols using the Friedman test with significance set at P<0.05. Significant differences in SUVs were observed in various part of the brain associated with each anesthetic protocol. The SUV for the frontal and occipital lobes was significantly higher than in the brainstem in all dogs. Dogs receiving medetomidine/tiletamine–zolazepam also had significantly higher whole brain SUVs than the propofol/isoflurane group. We concluded that each anesthetic protocol exerted a different regional brain glucose uptake pattern. As a result, when comparing brain glucose uptake using PET/CT, one should consider the effects of anesthetic protocols on different regions of the glucose uptake in the dog's brain.     

FDG‐PET IN HEALTHY AND EPILEPTIC LAGOTTO ROMAGNOLO DOGS AND CHANGES IN BRAIN GLUCOSE UPTAKE WITH AGE

Regional cerebral metabolism and blood flow can be measured noninvasively with positron emission tomography (PET). 2‐[¹⁸F]fluoro‐2‐deoxy‐D‐glucose (FDG) widely serves as a PET tracer in human patients with epilepsy to identify the seizure focus. The goal of this prospective study was to determine whether juvenile or adult dogs with focal‐onset epilepsy exhibit abnormal cerebral glucose uptake interictally and whether glucose uptake changes with age. We used FDG‐PET to examine six Lagotto Romagnolo dogs with juvenile epilepsy, two dogs with adult‐onset epilepsy, and five control dogs of the same breed at different ages. Three researchers unaware of dog clinical status visually analyzed co‐registered PET and magnetic resonance imaging (MRI) images. Results of the visual PET analyses were compared with electroencephalography (EEG) results. In semiquantitative analysis, relative standard uptake values (SUV) of regions of interest (ROI) drawn to different brain regions were compared between epileptic and control dogs. Visual analysis revealed areas of hypometabolism interictally in five out of six dogs with juvenile epilepsy in the occipital, temporal, and parietal cortex. Changes in EEG occurred in three of these dogs in the same areas where PET showed cortical hypometabolism. Visual analysis showed no abnormalities in cerebral glucose uptake in dogs with adult‐onset epilepsy. Semiquantitative analysis detected no differences between epileptic and control dogs. This result emphasizes the importance of visual analysis in FDG‐PET studies of epileptic dogs. A change in glucose uptake was also detected with age. Glucose uptake values increased between dog ages of 8 and 28 weeks and then remained constant.     

CHARACTERIZATION OF PHYSIOLOGIC 18F‐FDG UPTAKE WITH PET‐CT IN DOGS

We evaluated the whole body distribution of 2‐deoxy‐2‐[18F]fluoro‐d ‐glucose (¹⁸F‐FDG) in seven beagle dogs using positron emission tomography/computed tomography. The mean and maximum standard uptake values (SUV) for various tissues were computed. The SUV of the aortic blood pool was 0.65±0.19. Moderate uptake was present in brain (3.40±1.01). Mild uptake was present in orbital muscles, soft palate, laryngeal and pharyngeal region, mandibular salivary gland, myocardium, liver, pancreas, kidney, and intestine. ¹⁸F‐FDG uptake would be normally higher in these tissues because of normal physiologic activity. Mean and maximum SUV values of the eye, skeletal muscle, bone tissue, spleen, adrenal gland, stomach, tongue, gall bladder, and lung were similar to or lower than that of the aortic blood pool. These data provide a normal baseline for comparing pathologic ¹⁸F‐FDG uptake.     

COMPARISON BETWEEN 2‐18F‐FLUORO‐2‐DEOXY‐D‐GLUCOSE POSITRON EMISSION TOMOGRAPHY AND CONTRAST‐ENHANCED COMPUTED TOMOGRAPHY FOR MEASURING GROSS TUMOR VOLUME IN CATS WITH ORAL SQUAMOUS CELL CARCINOMA

Feline oral squamous cell carcinoma is one of the most refractory feline malignancies. Most patients succumb due to failure in local tumor control. 2‐¹⁸F‐fluoro‐2‐deoxy‐D‐glucose positron emission tomography (¹⁸F‐FDG PET) is increasingly being used for veterinary oncology staging as it highlights areas with higher glucose metabolism. The goal of the current prospective study was to compare gross tumor volume measurements using ¹⁸F‐FDG PET vs. those using computed tomography (CT) for stereotactic radiation therapy planning in cats with oral squamous cell carcinoma. Twelve cats with confirmed oral squamous cell carcinoma underwent pretreatment ¹⁸F‐FDG PET/CT. Gross tumor volumes based on contrast‐enhanced CT and ¹⁸F‐FDG PET were measured and compared among cats. Mean PET gross tumor volume was significantly smaller than mean CT gross tumor volume in the mandibular/maxillary squamous cell carcinoma group (n = 8, P = 0.002) and for the total number of patients (n = 12, P = 0.006), but not in the lingual/laryngeal group (n = 4, P = 0.57). Mismatch fraction analysis revealed that most of the lingual/laryngeal patients had a large region of high‐¹⁸F‐FDG activity outside of the CT gross tumor volume. This mismatch fraction was significantly greater in the lingual/laryngeal group than the mandibular/maxillary group (P = 0.028). The effect of poor spatial resolution of PET imaging was greater when the absolute tumor volume was small. Findings from this study indicated that ¹⁸F‐FDG PET warrants further investigation as a supplemental imaging modality in cats with oral squamous cell carcinoma because it detected regions of possible primary tumor that were not detected on CT images.     

POSITRON EMISSION TOMOGRAPHY OF THE EQUINE DISTAL LIMB: EXPLORATORY STUDY

Positron emission tomography (PET) is a highly sensitive, noninvasive imaging technique for quantifying biological functions of tissues. However, at the time of this study, PET imaging applications had not been reported in the horse. The aim of this exploratory study was to determine whether a portable high‐resolution PET scanner could be used to image the equine distal limb. Images of the front feet and fetlocks of three research horses, with known lesions localized to the distal front limbs, were acquired under general anesthesia after administration of ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG), with doses ranging from 1.5 to 2.9 MBq/kg. The radiation exposure measured during imaging was slightly higher than ^99mTechnetium scintigraphy. However, the use of general anesthesia allowed the proximity and the contact time with the patient to be minimized for the staff involved. ¹⁸F‐FDG uptake was evident throughout the soft tissues, with the highest uptake in the coronary band and the lowest uptake in the tendons. Anatomic structures could be discriminated due to the high contrast between soft tissue and bone. Detected lesions included lysis of the flexor cortex of the navicular bone, lesions of flexor tendons and suspensory ligament, and abnormal uptake through the lamina of a laminitic subject. Findings indicated that tomographic molecular imaging is feasible in the equine distal limb and could be useful as a future diagnostic technique for clinical and research studies, especially those involving tendinopathy/desmopathy and laminitis.     

Evaluation of 18F‐FDG PET/CT as a diagnostic imaging and staging tool for feline oral squamous cell carcinoma

18F‐fluorodeoxyglucose positron emission tomography combined with computed tomography (18FDG‐PET/CT) has been shown to be effective for staging human oral squamous cell carcinoma (SCC) but its application for cats with oral SCC is unknown. Twelve cats with biopsy‐proven oral SCC were imaged with whole body 18FDG‐PET/CT to determine its value as a diagnostic imaging and staging tool and fine needle aspirates were obtained of accessible regional lymph nodes. All tumors were FDG avid and conspicuous on 18FDG‐PET/CT images, with an average of the maximum standardized uptake value 9.88 ± 5.33 SD (range 2.9–24.9). Soft tissue infiltrative tumors that were subtle and ill defined on CT were highly visible and more extensive on FDG‐PET/CT. Tumors invading the osseous structures were more similar in extent on 18FDG‐PET/CT and CT although they were more conspicuous on PET images. Three cytologically confirmed metastases were hypermetabolic on PET, while two of those metastases were equivocal on CT.     

CEREBRAL GLUCOSE UTILIZATION MEASURED WITH HIGH RESOLUTION POSITRON EMISSION TOMOGRAPHY IN EPILEPTIC FINNISH SPITZ DOGS AND HEALTHY DOGS

In human epileptic patients, changes in cerebral glucose utilization can be detected 2‐deoxy‐2‐[¹⁸F] fluoro‐d ‐glucose positron emission tomography (FDG‐PET). The purpose of this prospective study was to determine whether epileptic dogs might show similar findings. Eleven Finnish Spitz dogs with focal idiopathic epilepsy and six healthy dogs were included. Dogs were examined using electroencephalography (EEG) and FDG‐PET, with epileptic dogs being evaluated during the interictal period. Visual and semi‐quantitative assessment methods of FDG‐PET were compared and contrasted with EEG findings. Three independent observers, unaware of dog clinical status, detected FDG‐PET uptake abnormalities in 9/11 epileptic (82%), and 4/8 healthy dogs (50%). Occipital cortex findings were significantly associated with epileptic status (P = 0.013). Epileptic dogs had significantly lower standardized uptake values (SUVs) in numerous cortical regions, the cerebellum, and the hippocampus compared to the control dogs. The lowest SUVs were found in the occipital lobe. White matter normalized and left‐right asymmetry index values for all pairs of homologous regions did not differ between groups. Visual evaluation of the EEGs was less sensitive (36%) than FDG‐PET. Both diagnostic tests were consensual and specific (100%) for occipital findings, but EEG had a lower sensitivity for detecting lateralized foci than FDG‐PET. Findings supported the use of FDG‐PET as a diagnostic test for dogs with suspected idiopathic epilepsy. Visual and semiquantitative analyses of FDG‐PET scans provided complementary information. Findings also supported the theory that epileptogenesis may occur in multiple brain regions in Finnish Spitz dogs with idiopathic epilepsy.     

POSITRON EMISSION TOMOGRAPHY FEATURES OF CANINE NECROTIZING MENINGOENCEPHALITIS

A Yorkshire terrier and a Chihuahua were referred for acute onset, generalized tonic‐clonic seizures and were suspected to have meningoencephalitis based on magnetic resonance (MR) imaging findings. Brain lesions appeared hyperintense with T2‐weighted imaging and hypointense with T1‐weighted imaging, and were characteristic of necrotizing meningoencephalitis. Both dogs were diagnosed with necrotizing meningoencephalitis based on pathologic findings. Fluorine‐18 fluorodeoxyglucose positron emission tomography (FDG‐PET) was performed on both animals before euthanasia with the permission of the owner. In FDG‐PET images, these lesions seen in MR images were characterized by multifocal or diffuse hypometabolism. Our FDG‐PET results provided evidence of glucose hypometabolism in areas of necrosis and cavitation associated with necrotizing meningoencephalitis. FDG‐PET has the potential to provide valuable diagnostic information in dogs with suspected necrotizing encephalitis.     

18F‐FDG‐PET/CT as adjunctive diagnostic modalities in canine fever of unknown origin

Fever of unknown origin (FUO) is a persistent or recurrent fever for which the underlying source has not been identified despite diagnostic investigation. In people, ¹⁸F‐fluoro‐2‐deoxyglucose positron emission tomography (¹⁸F‐FDG‐PET) alone or in combination with computed tomography (CT) is often beneficial in detecting the source of fever when other diagnostics have failed. Veterinary reports describing use of these modalities in animals with fever of unknown origin are currently lacking. Aims of this retrospective case series were to describe ¹⁸F‐FDG‐PET or ¹⁸F‐FDG‐PET/CT findings in a group of dogs with fever of unknown origin. Dogs presenting to a single center between April 2012 and August 2015 were included. A total of four dogs met inclusion criteria and underwent either positron emission tomography (n = 2) or positron emission tomography/CT (n = 2) as a part of their diagnostic investigation. All subjects underwent extensive diagnostic testing prior to ¹⁸F‐FDG‐PET/CT. Initial diagnostic evaluation failed to identify either a cause of fever or an anatomic location of disease in these four dogs. In each dog, positron emission tomography or positron emission tomography/CT was either able to localize or rule out the presence of focal lesion thereby allowing for directed sampling and/or informed disease treatment. Follow up ¹⁸F‐FDG‐PET/CT scans performed in two patients showed improvement of observed abnormalities (n = 1) or detected recurrence of disease allowing for repeated treatment before clinical signs recurred (n = 1). Fever resolved after specific treatment in each dog. Findings from the current study supported the use of positron emission tomography or positron emission tomography/CT as adjunctive imaging modalities for diagnosis and gauging response to therapy in dogs with fever of unknown origin.     

WHOLE‐BODY BIODISTRIBUTION OF 3′‐DEOXY‐3′‐[18F]FLUOROTHYMIDINE (18FLT) IN HEALTHY ADULT CATS

Positron emission tomography/computed tomography (PET/CT) utilizing 3′‐deoxy‐3′‐[¹⁸F]fluorothymidine (¹⁸FLT), a proliferation tracer, has been found to be a useful tool for characterizing neoplastic diseases and bone marrow function in humans. As PET and PET/CT imaging become increasingly available in veterinary medicine, knowledge of radiopharmaceutical biodistribution in veterinary species is needed for lesion interpretation in the clinical setting. The purpose of this study was to describe the normal biodistribution of ¹⁸FLT in adult domestic cats. Imaging of six healthy young adult castrated male cats was performed using a commercially available PET/CT scanner consisting of a 64‐slice helical CT scanner with an integrated whole‐body, high‐resolution lutetium oxy‐orthosilicate (LSO) PET scanner. Cats were sedated and injected intravenously with 108.60 ± 2.09 (mean ± SD) MBq of ¹⁸FLT (greater than 99% radiochemical purity by high‐performance liquid chromatography). Imaging was performed in sternal recumbency under general anesthesia. Static images utilizing multiple bed positions were acquired 80.83 ± 7.52 (mean ± SD) minutes post‐injection. Regions of interest were manually drawn over major parenchymal organs and selected areas of bone marrow and increased tracer uptake. Standardized uptake values were calculated. Notable areas of uptake included hematopoietic bone marrow, intestinal tract, and the urinary and hepatobiliary systems. No appreciable uptake was observed within brain, lung, myocardium, spleen, or skeletal muscle. Findings from this study can be used as baseline data for future studies of diseases in cats.     

STATIC AND DYNAMIC 18FDG‐PET IN NORMAL HISPANIOLAN AMAZON PARROTS (AMAZONA VENTRALIS)

Positron emission tomography (PET) is often used to stage and monitor human cancer and has recently been used in a similar fashion in veterinary medicine. The most commonly used radiopharmaceutical is 2‐Deoxy‐2‐[¹⁸F]‐Fluoro‐d ‐glucose (¹⁸F‐FDG), which is concentrated and trapped within cells that use glucose as their energy substrate. We characterized the normal distribution of ¹⁸F‐FDG in 10 healthy Hispaniolan Amazon parrots (Amazona ventralis) by performing whole body PET scans at steady state, 60 min after injection. Significant variability was found in the intestinal activity. Avian species are known to reflux fluid and electrolytes from their cloaca into their colon. To evaluate reflux as the cause of variability in intestinal distribution of ¹⁸F‐FDG, dynamic PET scans were performed on the coelomic cavity of six Hispaniolan Amazon parrots from time 0 to 60 min postinjection of radiotracer. Reflux of radioactive material from the cloaca into the colon occurred in all birds to varying degrees and occurred before 60 min. To evaluate the intestinal tract of clinical avian patients, dynamic scans must be performed starting immediately after injection so that increased radioactivity due to metabolism or hypermetabolic lesions such as cancer can be differentiated from increased radioactivity due to reflux of fluid from the cloaca.     

PET/CT today and tomorrow in veterinary cancer diagnosis and monitoring: fundamentals,early results and future perspectives

Functional imaging using positron emission tomography (PET) plays an important role in the diagnosis, staging, image‐guided treatment planning and monitoring of malignant diseases. PET imaging complements conventional anatomical imaging such as computed tomography (CT) and magnetic resonance imaging (MRI). The strength of CT scanning lies in its high spatial resolution, allowing for anatomical characterization of disease. PET imaging, however, moves beyond anatomy and characterizes tissue based on functions such as metabolic rate. Combined PET/CT scanners were introduced commercially in 2001 and a number of technological advancements have since occurred. Radiolabelled tracers such as ¹⁸F‐fluorodeoxyglucose (FDG) and ¹⁸F‐fluorothymidine (FLT) allow visualization of various metabolic processes within cancer cells. Many studies in human oncology evaluating the utility of PET/CT have demonstrated clinical benefits. Few veterinary studies have been performed, but initial studies show promise for improved detection of malignancy, more thorough staging of canine cancer and determination of early response and disease recrudescence.     

PHYSIOLOGIC VARIANTS,BENIGN PROCESSES,AND ARTIFACTS FROM 106 CANINE AND FELINE FDG‐PET/COMPUTED TOMOGRAPHY SCANS

18F‐Fluoro‐deoxyglucose positron emission computed tomography (FDG‐PET/CT) is an emerging diagnostic imaging modality in veterinary medicine; however, little published information is available on physiologic variants, benign processes, and artifacts. The purpose of this retrospective study was to describe the number of occurrences of non‐neoplastic disease‐related FDG‐PET/CT lesions in a group of dogs and cats. Archived FDG‐PET/CT scans were retrieved and interpreted based on a consensus opinion of two board‐certified veterinary radiologists. Non‐neoplastic disease‐related lesions were categorized as physiologic variant, benign activity, or equipment/technology related artifact. If the exact cause of hypermetabolic areas could not be determined, lesions were put into an indeterminate category. A total of 106 canine and feline FDG‐PET/CT scans were included in the study. In 104 of the 106 scans, a total of 718 occurrences of physiologic variant, areas of incidental benign activity, and artifacts were identified. Twenty‐two of 23 feline scans and 82 of 83 canine scans had at least one artifact. Previously unreported areas of increased radiopharmaceutical uptake included foci associated with the canine gall bladder, linear uptake along the canine mandible, and focal uptake in the gastrointestinal tract. Benign activity was often seen and related to healing, inflammation, and indwelling implants. Artifacts were most often related to injection or misregistration. Further experience in recognizing the common veterinary FDG physiologic variation, incidental radiopharmaceutical uptake, and artifacts is important to avoid misinterpretation and false‐positive diagnoses.     

IMAGING DIAGNOSIS—MULTICENTRIC LYMPHOMA OF GRANULAR LYMPHOCYTES IMAGED WITH FDG PET/CT IN A DOG

A 5‐year‐old female spayed bulldog was referred for mild dyspnea, decreased activity and appetite, occasional nonproductive cough, polydipsia, and polyuria. A 2‐deoxy‐2‐[¹⁸F]fluoro‐D‐glucose (FDG) positron emission tomography/computed tomography (PET/CT) scan revealed unexpected activity in the heart, lungs, and mild generalized lymphadenopathy that led to the diagnosis of lymphoma of granular lymphocytes after nonspecific findings on imaging with standard modalities of echocardiography, thoracic radiography, and abdominal ultrasound. PET/CT scanning is a useful whole body imaging modality with high sensitivity for changes associated with canine lymphoma.     

Pilot study utilizing Fluorine‐18 fluorodeoxyglucose‐positron emission tomography/computed tomography for glycolytic phenotyping of canine mast cell tumors

The goal of this prospective pilot study was to use naturally occurring canine mast cell tumors of various grades and stages as a model for attempting to determine how glucose uptake and markers of biologic behavior are correlated. It was hypothesized that enhanced glucose uptake, as measured by 2‐[fluorine‐18]fluoro‐d ‐glucose‐positron emission tomography/computed tomography (F18 FDG PET‐CT), would correlate with histologic grade. Dogs were recruited for this study from a population referred for treatment of cytologically or histologically confirmed mast cell tumors. Patients were staged utilizing standard of care methods (abdominal ultrasound and three view thoracic radiographs), followed by a whole body F18 FDG PET‐CT. Results of the F18 FDG PET‐CT were analyzed for possible metastasis and standard uptake value maximum (SUV_max) of identified lesions. Incisional or excisional biopsies of the accessible mast cell tumors were obtained and histology performed. Results were then analyzed to look for a possible correlation between the grade of mast cell tumors and SUV_max. A total of nine animals were included in the sample. Findings indicated that there was a correlation between grade of mast cell tumors and SUV_max as determined by F18 FDG PET‐CT (p‐value = 0.073, significance ≤ 0.1). Based on the limited power of this study, it is felt that further research to examine the relationship between glucose utilization and biologic aggressiveness in canine mast cell tumors is warranted. This study was unable to show that F18 FDG PET‐CT was a better staging tool than standard of care methods.     

The effect of medetomidine on the regional cerebral blood flow in dogs measured using Technetium-99m-Ethyl Cysteinate Dimer SPECT

Sedatives and anaesthetics are known to cause changes in the regional cerebral blood flow. In dogs intramuscular sedation with medetomidine, a potent sedative frequently used in veterinary medicine, is sometimes indicated prior to intravenous injection of ^99mTechnetium-Ethyl Cysteinate Dimer (^99mTc-ECD) in brain perfusion studies using Single Photon Emission Computed Tomography (SPECT). Based on the knowledge of the distribution of alpha₂-receptors in the brain, we hypothesized altered regional brain perfusion in dogs receiving medetomidine prior to ^99mTc-ECD. Two conditions were compared in 10 dogs; tracer injection before and after intramuscular sedation with medetomidine. In our study, medetomidine caused a significantly higher tracer uptake in all brain regions. Semi-quantification of brain perfusion rendered a lower perfusion index in the subcortical region and an imbalance between left and right cortical perfusion induced by medetomidine. This study shows that caution is needed when quantifying the brain perfusion indices under medetomidine sedation.     

The fine structure of the Harderian gland in the ostrich (Struthio camelus)

The Harderian gland of the ostrich (Struthio camelus) is a tubuloalveolar gland containing holocrine secreting epithelial cells. The gland epithelium is composed of two different cell types, which can be classified as type I and type II. These cells contain dense secretory vesicles in their cytoplasm and they are connected laterally with desmosomes. At the basal site of these cells, myoepithelial cells are present. Plasma cells are observed in the subepithelial region of the gland. In the interlobular trabeculae, forming the gland stroma, fibroblasts, blood vessels and nerve fibres are included. Another important finding in the ostrich Harderian gland is the presence of homogeneous material.     

Harderian gland neoplasms in captive, wild-caught Beechey ground squirrels (Spermophilus beecheyi)

Harderian gland neoplasms were identified in 18 aged, adult Beechey ground squirrels (Spermophilus beecheyi) from the records of 167 wild-caught captive animals that were necropsied. All but one animal had tumors that were classified as carcinomas, with infiltrative growth and frequent metastases. This is the first detailed report of Harderian gland neoplasia in wild Sciuridae, although this neoplasm has been described in other rodent species. Clinically, affected ground squirrels typically were inappetent and presented with weight loss and exophthalmos. The biologic behavior of Harderian gland neoplasia is variable among rodent species; in Beechey ground squirrels there was a high incidence of malignant behavior. Eleven of 17 tumor-bearing animals for which the gender was known were male, and 6 were female. Nine of 16 for which data were available were uninfected, and 7 had evidence of current or prior infection with ground squirrel hepatitis virus. Tumor development occurred in older animals; all but 2 were 5.5 years of age or older. The presence of metastasis was not related to gender or chronic ground squirrel hepatitis virus infection.