Detecting and quantifying marijuana metabolites in serum and urine of 19 dogs affected by marijuana toxicity.

Veterinarians diagnose marijuana toxicity based on clinical signs and history, or in conjunction with an over-the-counter (OTC) human urine drug screen. With the legalization of recreational marijuana use becoming more prevalent in the United States, a more accurate test to aid in the diagnosis of canine marijuana toxicity is needed. We collected urine and serum samples from 19 dogs with confirmed or suspected marijuana toxicosis from multiple veterinary hospitals and analyzed them with a novel UPLC-MS/MS method. Calibrations from 0.1 to 100 ng/mL and QC materials were prepared. Samples were extracted, purified, and eluted with solid-phase extraction. Urine samples were tested with an OTC human urine drug screen. The limit of detection (LOD) and lower limit of quantification (LLOQ) ranges for marijuana metabolites in serum were 0.05-0.25 ng/mL and 0.1-0.5 ng/mL, respectively. In urine, the LOD and LLOQ ranges for the metabolites were 0.05-0.1 ng/mL and 0.1-0.5 ng/mL, respectively. In serum, median and range of metabolite concentrations (ng/mL) detected included: THC, 65.0 (0.14-160); 11-OH-Δ9-THC, 4.78 (1.15-17.8); 11-nor-9-carboxy-Δ9-THC, 2.18 (0.71-7.79); CBD, 0.28 (0.11-82.5); and THC-glucuronide, 2.05 (0.72-18.3). In the 19 urine samples, metabolite: creatinine (ng: mg) values detected included: THC, 0.22 (0.05-0.74); 11-OH-Δ9-THC, 0; 11-nor-9-carboxy-Δ9-THC, 1.32 (0.16-11.2); CBD, 0.19 (0.12-0.26); THC-COOH-glucuronide, 0.08 (0.04-0.11); and THC-glucuronide, 0.98 (0.25-10.7). Twenty of 21 urine samples tested negative for THC on the urine drug screen. All 19 serum samples contained quantifiable concentrations of THC using our novel UPLC-MS/MS method. Utilizing a UPLC-MS/MS method can be a useful aid in the diagnosis of marijuana toxicosis in dogs, whereas using an OTC human urine drug test is not a useful test for confirming marijuana exposure in dogs because of the low concentration of THC-COOH in urine.

REFRACTOMETRIC URINE SPECIFIC GRAVITY OF FREE-LIVING EGYPTIAN FRUIT BATS (ROUSETTUS AEGYPTIACUS).

In both human and veterinary medicine, urine specific gravity (USG) is commonly measured by refractometry to indirectly reflect the osmolality of urine to thereby evaluate the kidney's ability to concentrate or dilute urine according to physiologic need and certain disease conditions. However, for accurate interpretation of the significance of any value, knowledge of the expected USG for the healthy species in question is required. It is generally believed that fruit bats, and Egyptian fruit bats (Rousettus aegyptiacus) in particular, are unable to highly concentrate their urine. In this study, the USG was determined using a handheld urine refractometer in 43 free-living Egyptian fruit bats of both sexes. The calculated nonparametric 90% confidence interval for Egyptian fruit bats in this study was 1.006-1.050, with no association with capture site, sex, weight, or packed cell volume and total solids. Results suggest that free-living Egyptian fruit bats are able to highly concentrate their urine.

PLASMA THYROXINE (T4) CONCENTRATION IN ZOO-KEPT BLACK-TAILED PRAIRIE DOGS (CYNOMYS LUDOVICIANUS).

This study was conducted to determine plasma thyroxine (T4) concentrations in zoo-kept black-tailed prairie dogs ( Cynomys ludovicianus ). Thirty-one healthy prairie dogs of both sexes were studied as part of their annual clinical health evaluation, performed under general isoflurane anesthesia. Each animal underwent a complete physical examination, complete blood count, plasma biochemistry, and venous blood gas analysis. Heparinized venous blood samples were collected individually and processed for plasma T4 analysis using a veterinary biochemistry analyzer. The median plasma T4 concentration for the prairie dogs in this study was 4.1 µg/dl (minimum = 0.6 µg/dl; maximum = 8.0 µg/dl). The mean ± standard deviation plasma T4 concentration was 4.49 ± 2.39 µg/dl. No significant differences were found for varying ages, sexes, weights, or housing systems. The data presented in this report can promote better physiologic understanding and improve clinical management of this rodent species.

Stability of osmolality in previously frozen canine serum and urine samples.

BACKGROUND: The measurement of osmolality is of interest in several clinical conditions and has been investigated in multiple veterinary studies. However, the stability of osmolality over time in frozen canine serum and urine has not been established. OBJECTIVES: The purpose of this study was to determine the stability of osmolality in canine serum and urine stored frozen at -20°C and -80°C up to 90 days, and to assess the effect of storage temperature on osmolality measurement. METHODS: Serum and urine samples collected from 5 healthy Greyhound dogs were aliquoted and stored at -20°C and -80°C until measurement. Osmolality, assessed by freezing-point depression, was measured at 0, 7, 14, 30, and 90 days of storage. RESULTS: For both serum and urine, osmolality at day 7 was not statistically different from day 0, but osmolality at days 14, 30, and 90 was significantly lower than at day 0. There was no significant effect of storage temperature on serum osmolality. However, the osmolality of urine samples stored at -20°C was slightly, but significantly lower than the osmolality of those stored at -80°C. CONCLUSIONS: Osmolality measurement in the serum and urine of healthy dogs appears to be stable for at least 7 days in frozen samples. The small changes observed after day 7 are unlikely to be relevant for individual patients, but could have implications in research study protocols.

SELECTED PLASMA BIOCHEMISTRY ANALYTES OF HEALTHY CAPTIVE SULCATA (AFRICAN SPURRED) TORTOISES (CENTROCHELYS SULCATA).

The sulcata or African spurred tortoise (Centrochelys sulcata) is a large tortoise species that is commonly kept in zoologic collections and as a pet. The objectives of this study were to establish reference intervals for selected biochemical analytes in clinically healthy captive sulcata tortoises and to evaluate the impact of blood sampling site and sex. Blood samples were collected from 60 tortoises from either the dorsal coccygeal (tail) vein or the subcarapacial venous plexus based on their body size. The packed cell volume and refractometric total solids (TS) were determined. The concentrations of selected plasma biochemical analytes were determined using the VetScan VS2 analyzer and included albumin, aspartate aminotransferase, bile acids, calcium, creatine kinase, globulins, glucose, potassium, sodium, phosphorous, total proteins (TP), and uric acid. The calcium-to-phosphorous ratio was calculated. Reference intervals were determined and evaluated for the potential effects of blood sampling site and sex. There were significant differences (P < 0.05) associated with the blood sampling site in TS, TP, phosphorus, and globulins, with higher values in samples from the tail versus the subcarapacial sampling site. No significant statistical differences were noted in the plasma biochemistry analytes between the sexes. Reading of the globulins by the analyzer failed in 36 of 60 of the samples and was largely associated with the subcarapacial plexus sampling site. The reference intervals defined by the VetScan analyzer in this study can be used for clinical medicine and conservation of this tortoise species. Sampling site was identified as a factor significantly affecting some blood analytes in this study; this factor should thus be taken into consideration when assessing sulcata tortoise health status and using this testing methodology.

Regional Nodal Metastasis of Humeral Chondrosarcoma in a Dog.

A 6 yr old castrated male English springer spaniel was evaluated with a 1 mo history of progressive right forelimb lameness with recent swelling around the elbow joint. Physical examination findings included lameness of the right forelimb, muscle atrophy around the right shoulder, grade 2/6 heart murmur, and moderate dental disease. Results of a complete blood cell count and serum biochemical analysis were unremarkable with the exception of a mildly increased alkaline phosphatase (368 U/L; reference range, 128-328 U/L). Radiographs of the right elbow revealed a mixed lytic and proliferative osseous lesion most consistent with either neoplasia or infection. Thoracic radiographs and the echocardiogram were unremarkable. Fine-needle aspiration of the bone lesion was performed. The cytological diagnosis was chondrosarcoma. The right forelimb was amputated and the axillary lymph nodes were collected. Histopathological examination of the bone lesion and axillary lymph nodes revealed chondrosarcoma with metastasis to the lymph nodes. Lymph node metastasis of chondrosarcoma is rare and needs to be further evaluated as a prognostic indicator.

Evaluation of mean corpuscular volume difference as a marker for serum hypertonicity during water deprivation in dogs.

OBJECTIVE: To evaluate mean corpuscular volume difference (dMCV) as a marker for hypertonicity induced by water deprivation in dogs. ANIMALS: 5 healthy Greyhounds maintained in a research colony. PROCEDURES: Water was withheld for 24 hours. Blood and urine samples were collected before (time 0) and every 6 hours during water deprivation. Serum and urine osmolality were measured on the basis of freezing point depression, and dMCV was calculated from routine hematologic variables. RESULTS: Serum and urine osmolality significantly increased and body weight decreased over time in healthy Greyhounds during water deprivation, although most dogs developed only a slight increase in serum osmolality. The dMCV also increased over time, but the value at 24 hours did not differ significantly from the value at time 0. However, a significant correlation was found between serum osmolality and dMCV. A dMCV ≥ 5 fL yielded 100% specificity for predicting hypertonicity when hypertonicity was defined as serum osmolality ≥ 310 mOsM. CONCLUSIONS AND CLINICAL RELEVANCE: dMCV may be a useful marker for detection of mild hypertonicity in dogs and may have clinical and research applications for use in screening canine populations for hypertonicity.

Splenopancreatic ductal adenocarcinoma with multiorgan metastasis in a northern water snake (Nerodia sipedon).

A 16-yr-old northern water snake (Nerodia sipedon) presented with a large, firm midcoelomic swelling. A complete blood count, survey radiographs, coelomic ultrasound, and fine needle aspirate of the mass were performed. Survey radiographs revealed a soft tissue opacity mass. Ultrasonographic examination confirmed the presence of the mass but could not aid in its visceral localization. After 2 weeks, the snake presented again because of continued anorexia and poor quality of life. Euthanasia was performed. Gross necropsy revealed a multilobulated mass attached to and effacing the splenopancreas. Histologically, the mass was composed of cuboidal to columnar neoplastic epithelial cells forming tubules surrounded by variable amounts of fibrovascular stroma. Histological examination and immunohistochemical staining of other tissues revealed local invasion in the subserosa and tunica muscularis of the stomach, metastasis within the liver, in the mesovarium, and an intravascular metastasis within the ventricle of the heart surrounded by a thrombus.

In vitro increase of mean corpuscular volume difference (dMCV) as a marker for serum hypertonicity in dogs.

Spurious increase in erythrocyte mean corpuscular volume (MCV) on automated cell analyzers is a well-characterized lab error in hypertonic patients. A difference between automated and manual MCV (dMCV) greater than 2 fl has been shown to predict hypertonicity in humans. The purpose of this study was to investigate dMCV as a marker for serum hypertonicity in dogs and to examine the relationship between dMCV and three methods of estimating serum tonicity: measured (OsMM), calculated (OsMC), and calculated effective (OsMCE) osmolalities. OsMC, OsMCE, and dMCV were calculated from routine blood values and OsMM was directly measured in 121 dogs. The dMCV of hypertonic dogs was significantly larger than that of normotonic dogs for all three osmolality methods. dMCV predicted hypertonicity as estimated by OsMM better than it predicted hypertonicity as estimated by OsMC and OsMCE. A cutoff of 2.96 fl yielded the best sensitivity (76%) and specificity (71%) for hypertonicity estimated by OsMM.

Peritoneal EMH in a dog with immune-mediated hemolytic anemia.

Extramedullary hematopoiesis (EMH) is the process by which normal blood cells are produced outside the bone marrow. In humans, EMH effusions are rare and are characterized by the presence of megakaryocytes, immature erythrocytes, immature leukocytes, or combinations of those cells. To the authors' knowledge, this is the first report to describe a case of peritoneal EMH effusion in a dog. A 5 yr old castrated male shorthaired dachshund presented with a 2 day history of pigmenturia and inappetence. A complete blood count revealed regenerative anemia with marked agglutination, spherocytosis, and an acute inflammatory leukogram characterized by a neutrophilia, regenerative left shift, and monocytosis. Ultrasound-guided aspiration of peritoneal effusion yielded a sample of high nucleated cellularity predominantly composed of mature and immature neutrophils and erythroid precursor cells. The patient was diagnosed with primary immune-mediated hemolytic anemia with concurrent EMH peritoneal effusion. The following case description and discussion explore the clinical findings associated with the unusual effusion and outline the possible pathogenesis by which the EMH effusion may have arisen in the dog.

Severe anemia caused by babesiosis in a maned wolf (Chrysocyon brachyurus).

An 8-yr-old, captive, spayed, female maned wolf (Chrysocyon brachyurus) developed progressive lethargy and weakness over a 24-hr period. Clinical signs included vomiting, recumbency, horizontal nystagmus, possible blindness, pale icteric mucus membranes, and port-wine colored urine. A complete blood cell count revealed severe anemia (packed cell volume [PCV], 6%) and intraerythrocytic piroplasms consistent with a Babesia species. Polymerase chain reaction testing later confirmed babesiosis. The wolf was treated with imidocarb dipropionate, antibiotics, and fluid therapy. A whole-blood transfusion from a sibling maned wolf also was performed. Despite aggressive treatment, the wolf failed to improve and was euthanized. To the authors' knowledge, this is the first documented case of babesiosis in a captive maned wolf in North America. Surveillance of infectious diseases in captive and wild maned wolf populations should be expanded to include screening for Babesia species. Tick control also should be implemented to prevent and decrease transmission of the disease to this endangered species.