Diagnostic utility of the total nucleated cell count for differentiation of septic and sterile peritoneal effusions in dogs.

BACKGROUND: Rapid and accurate diagnosis of septic peritonitis is critical for initiating appropriate medical and surgical management. OBJECTIVES: The aim of this study was to determine the diagnostic utility of the total nucleated cell count (TNCC), absolute neutrophil count, neutrophil percentage, and total protein (TP) to distinguish septic versus non-septic peritoneal effusions in dogs. METHODS: Electronic medical records were retrospectively searched for peritoneal fluid samples from 2008 to 2018 and classified as septic or non-septic based on bacterial culture and/or cytology results. Receiver operator characteristic curves (ROCs) were used to describe the overall diagnostic utility of each test, with optimal cutpoints analyzed to dichotomize continuous variables. Positive and negative likelihood ratios were calculated at these cutpoints. RESULTS: A total of 166 unique samples, including 87 septic and 79 non-septic peritoneal effusions, were included. There were no significant differences in dog sex, age, or days hospitalized between groups. Septic effusions had significantly higher TP, TNCC, absolute neutrophil count, and neutrophil percentage compared with non-septic effusions. The area under the curve of the ROC curves was TNCC (0.80), absolute neutrophil count (0.80), neutrophil percentage (0.64), and TP (0.63). For TNCC and absolute neutrophil count, optimal cutoffs were 17.13 × 103 cells/µL and 19.88 × 103 cells/µL, resulting in positive and negative likelihood ratios of 2.39 and 0.28 and 2.85 and 0.28, respectively. CONCLUSIONS: Total nucleated cell counts and absolute neutrophil counts aid in the differentiation of septic and non-septic peritoneal effusions with similar diagnostic utility but are not sufficiently sensitive or specific to use without concurrent microscopic evaluation.

Osteosarcoma of the wing in a sulfur-crested cockatoo.

A 26-year-old female sulfur-crested cockatoo (Cacatua galerita) was evaluated for vocalizing through the night and extending her right wing. Physical examination revealed a large, firm mass extending from the humerus to the distal aspect of the elbow. Computed tomography confirmed a large aggressive mass of the right distal humerus with a large soft tissue component, severe osteolysis, and adjacent periosteal proliferation. Fine-needle aspirates of the mass were most compatible with sarcoma, and osteosarcoma was prioritized. An unstained slide was treated with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase (NBT/BCIP) substrate for ALP detection and was strongly positive, confirming a diagnosis of osteosarcoma. A month later, the patient underwent wing amputation and arrested during recovery from anesthesia. Post-mortem examination and histopathology were consistent with osteosarcoma. This case report highlights a rare occurrence of osteosarcoma in a cockatoo as well as its cytologic and histologic features. Additionally, this report provides support for NBT/BCIP application in ALP-expressing tumors, a cytochemical stain that has been minimally investigated in avian species.

Eosinophilic cavitary effusions in cats: 48 cases (2010-2020).

BACKGROUND: Eosinophilic effusions are commonly defined as effusions with ≥10% eosinophils. Eosinophilic cavitary effusions are infrequently observed in cats, with few case reports comprising the majority of the recent literature. OBJECTIVE: The objective was to review disease associations of cats with eosinophilic cavitary effusions and to assess if a lower threshold of eosinophils (5%-9%) may warrant consideration of similar etiologies associated with effusions with ≥10% eosinophils. METHODS: Cytology reports were retrospectively reviewed for all feline cavitary effusions submitted for fluid analysis from 2010 to 2020 at a veterinary teaching hospital. Cases were included if the manual leukocyte differential included ≥5% eosinophils and separated into 5%-9% and ≥10% eosinophils groups. Patient records were reviewed for associated medical conditions. RESULTS: A total of 669 effusions were submitted from 579 cats; 50 effusions from 48 cats had a leukocyte differential with ≥5% eosinophils. The eosinophil proportion was ≥10% in 22 cats; the most common underlying cause was neoplasia (10/22, 45%), followed by inflammatory disease (4/22, 18%), cardiac disease (3/22, 14%), suspect neoplasia (3/22, 14%), and undetermined (2/22, 9%). The underlying causes for the 26 cats with 5%-9% eosinophils were similar; neoplasia (8/26, 31%), cardiac disease (6/26, 23%), inflammatory disease (4/26, 15%), suspect neoplasia (3/26, 12%), undetermined (3/26, 12%), and idiopathic chylothorax (2/26, 8%). Cats with eosinophil proportions ≥10% in the fluid exhibited peripheral eosinophilia more frequently (35%) compared to those with 5%-9% eosinophils (5%). CONCLUSIONS: Consistent with the current literature, neoplasia, particularly lymphoma, remains a primary consideration for cats with eosinophilic effusions. Previously unreported associated diseases included cardiovascular and inflammatory disorders. Our findings suggest an eosinophil differential of 5%-9% is seen with similar etiologies considered for classically defined eosinophilic effusions.

Microbiome analysis of bile from apparently healthy cats and cats with suspected hepatobiliary disease.

BACKGROUND: Bacterial infection of bile is a common cause of hepatobiliary disease in cats. Whether bile harbors a core microbiota in health or in cases of suspected hepatobiliary disease in cats is unknown. OBJECTIVES: Establish if gallbladder bile in apparently healthy cats harbors a core microbiota composed of bacterial taxa common to many individuals. Compare results of bile cytology, bile culture, and 16S rRNA gene amplicon sequencing in apparently healthy cats and cats with suspected hepatobiliary disease. ANIMALS: Forty-three client-owned cats with suspected hepatobiliary disease and 17 control cats. METHODS: Bile was collected by ultrasound guided cholecystocentesis (cats with suspected hepatobiliary disease) or laparotomy after euthanasia (controls). Bile samples underwent cytologic examination, aerobic and anaerobic culture, and DNA was extracted for 16S rRNA gene amplification and sequencing. RESULTS: Microbiome sequencing did not identify a core microbiota in control cats or cats having bile sampled because of clinical suspicion for hepatobiliary disease. Microbiome profiles from control cats were indistinguishable from profiles obtained from sampling instruments and reagents that were not exposed to bile (technical controls). Bacterial taxa that could not be explained by contamination or off-target amplification were identified only in samples from cats with bactibilia and positive bile culture results for Escherichia coli. In several E. coli positive samples, microbiome sequencing also identified a small number of potentially co-infecting bacterial genera not identified by culture. CONCLUSIONS AND CLINICAL IMPORTANCE: Cat bile does not harbor a core microbiota. Uncultured bacteria may contribute to pathogenesis of hepatobiliary disease in cats with bile E. coli infection.

Gallbladder microbiota in healthy dogs and dogs with mucocele formation.

To date studies have not investigated the culture-independent microbiome of bile from dogs, a species where aseptic collection of bile under ultrasound guidance is somewhat routine. Despite frequent collection of bile for culture-based diagnosis of bacterial cholecystitis, it is unknown whether bile from healthy dogs harbors uncultivable bacteria or a core microbiota. The answer to this question is critical to understanding the pathogenesis of biliary infection and as a baseline to exploration of other biliary diseases in dogs where uncultivable bacteria could play a pathogenic role. A pressing example of such a disease would be gallbladder mucocele formation in dogs. This prevalent and deadly condition is characterized by excessive secretion of abnormal mucus by the gallbladder epithelium that can eventually lead to rupture of the gallbladder or obstruction of bile flow. The cause of mucocele formation is unknown as is whether uncultivable, and therefore unrecognized, bacteria play any systematic role in pathogenesis. In this study we applied next-generation 16S rRNA gene sequencing to identify the culture-negative bacterial community of gallbladder bile from healthy dogs and gallbladder mucus from dogs with mucocele formation. Integral to our study was the use of 2 separate DNA isolations on each sample using different extraction methods and sequencing of negative control samples enabling recognition and curation of contaminating sequences. Microbiota findings were validated by simultaneous culture-based identification, cytological examination of bile, and fluorescence in-situ hybridization (FISH) performed on gallbladder mucosa. Using culture-dependent, cytological, FISH, and 16S rRNA sequencing approaches, results of our study do not support existence of a core microbiome in the bile of healthy dogs or gallbladder mucus from dogs with mucocele formation. Our findings further document how contaminating sequences can significantly contribute to the results of sequencing analysis when performed on samples with low bacterial biomass.

A Platelet-Rich Plasma-Derived Biologic Clears Staphylococcus aureus Biofilms While Mitigating Cartilage Degeneration and Joint Inflammation in a Clinically Relevant Large Animal Infectious Arthritis Model.

The leading cause of treatment failure in Staphylococcus aureus infections is the development of biofilms. Biofilms are highly tolerant to conventional antibiotics which were developed against planktonic cells. Consequently, there is a lack of antibiofilm agents in the antibiotic development pipeline. To address this problem, we developed a platelet-rich plasma (PRP)-derived biologic, termed BIO-PLY (for the BIOactive fraction of Platelet-rich plasma LYsate) which has potent in vitro bactericidal activity against S. aureus synovial fluid free-floating biofilm aggregates. Additional in vitro studies using equine synoviocytes and chondrocytes showed that BIO-PLY protected these cells of the joint from inflammation. The goal of this study was to test BIO-PLY for in vivo efficacy using an equine model of infectious arthritis. We found that horses experimentally infected with S. aureus and subsequently treated with BIO-PLY combined with the antibiotic amikacin (AMK) had decreased bacterial concentrations within both synovial fluid and synovial tissue and exhibited lower systemic and local inflammatory scores compared to horses treated with AMK alone. Most importantly, AMK+BIO-PLY treatment reduced the loss of infection-associated cartilage proteoglycan content in articular cartilage and decreased synovial tissue fibrosis and inflammation. Our results demonstrate the in vivo efficacy of AMK+BIO-PLY and represents a new approach to restore and potentiate antimicrobial activity against synovial fluid biofilms.

Implementation of a Fine Needle Aspirate Simulation Model.

Being able to appropriately perform fine needle aspiration (FNA) collecting techniques and sample preparation is essential in obtaining a diagnostic sample, which is a critical skill for veterinary practitioners. Collection and preparation of cytologic samples are skills gained through practice. Experience leads to refinement of technique and improved diagnostic quality. Using live patients for mass skills training is not feasible; therefore, an aspiration simulation model and laboratory session was developed to reinforce physical exam skills, appropriate selection of sample collection supplies, and collection technique. Materials for the models include Ping-Pong balls, silicone, instant vanilla pudding mix, water, and stuffed animals. The laboratory session allows veterinary students to practice lesion identification, isolation, aspiration, and successful preparation. Subsequent submission of the collected sample involves being able to expel and spread the sample on a slide and proper labeling. While the simulation experience was initially developed for a short course with 12 students, it has recently been incorporated into the required clinical pathology clinical year rotation for up to 100 fourth-year veterinary students. The model is inexpensive and efficient and allows for technique development and immediate instructor assessment and feedback.

Hematologic and clinical characteristics of dogs with circulating macrophage-like cells.

BACKGROUND: Macrophage-like (ML) cells are rarely observed on blood smear examinations, and the significance of these cells has been poorly described. OBJECTIVE: The objective of this study was to retrospectively describe selected hematologic and clinical characteristics of dogs with ML cells in peripheral blood. MATERIALS AND METHODS: Complete blood count (CBC) reports with blood smear evaluations from the clinical pathology laboratory records at North Carolina University College of Veterinary Medicine were retrospectively reviewed. Data were collected over a 10-year-period. Dogs were defined as having circulating ML cells if three or more ML cells were present on a single blood smear. Hematologic and clinical data of dogs with circulating ML cells were compared with age-matched hospital-derived control dogs. RESULTS: Of 61,631 CBC records, 87 reports (0.14%) described the presence of ML cells. Thirty-nine dogs met the inclusion criteria. The hemogram of dogs with circulating ML cells was characterized by a pronounced inflammatory and stress leukogram, anemia, and thrombocytopenia. Of the 39 dogs, 19 (49%) had systemic or severe localized inflammatory/necrotic diseases. Eighteen (46%) dogs were diagnosed with neoplasia of histiocytic (5) and non-histiocytic origins (13). Dogs with circulating ML cells had a shorter median survival time (34 days) than the control dogs (595 days, P < .0001), with an increased occurrence of death/euthanasia within 1 month (3.89-fold). However, the presence of circulating ML cells was not found to be an independent prognostic factor in a multivariable model. CONCLUSIONS: The hemograms and diagnoses of dogs with ML cells suggest that severe inflammatory conditions or histiocytic and non-histiocytic neoplasia are common causes for circulating ML cells.

Factors associated with clinical interpretation of tracheal wash fluid from dogs with respiratory disease: 281 cases (2012-2017).

BACKGROUND: Clinicians face several dilemmas regarding tracheal washes (TWs) for the diagnosis of respiratory disease, including method and prediction of bacterial growth from cytology results. OBJECTIVE: To compare cytology and culture of endotracheal and transtracheal washes and identify factors associated with discordancy and bacterial growth. ANIMALS: Two hundred forty-five dogs with respiratory disease. METHODS: Retrospective study. Tracheal wash submissions were included if cellularity was sufficient for cytologic interpretation and aerobic cultures were performed. Collection technique, cytology, bacterial growth, and antibiotic history were analyzed. RESULTS: Fewer transtracheal specimens (9/144, 6.3%) were excluded for hypocellularity than endotracheal (28/174, 16.1%); otherwise, results were similar and were combined. Of 281 specimens with cellularity sufficient for interpretation, 97 (34.5%) had bacteria on cytology and 191 (68.0%) had bacterial growth. Cytology positive/culture negative discordancy was uncommon (8/97, 8%). Cytology negative/culture positive discordancy was frequent (102/184, 55.4%), but occurred less often (28/184, 14.2%) when only 1+ growth or greater was considered positive. Oropharyngeal contamination was associated with bacterial growth, but not discordancy. No association was found between antibiotic administration and bacterial growth. CONCLUSIONS AND CLINICAL IMPORTANCE: Endotracheal wash fluid, in particular, should be screened for gross mucus or turbidity to maximize the likelihood of an adequate specimen. Otherwise, endotracheal and transtracheal specimens were similar. Presence of bacteria on cytology was a good predictor of any growth, while their absence was a good predictor of the absence of growth of 1+ or more. Recent antibiotic usage should not discourage TW culture if there is compelling reason to avoid delay.

Evaluation of Retention of Veterinary Clinical Pathology Knowledge between Second-Year and Fourth-Year Clinical Pathology Courses.

There is a concern over long-term retention of knowledge in professional programs. The goal of this study was to evaluate the retention of veterinary clinical pathology knowledge between the fourth-semester and fourth-year clinical pathology courses. We hypothesize that students will forget a significant amount of content area knowledge between the fourth semester and fourth year in the Doctor of Veterinary Medicine (DVM) program. We further hypothesize that a review of material during the fourth-year clinical pathology rotation will help students rebuild existing knowledge and increase performance on specific test questions, between T2 (rotation pre-test) and T3 (rotation post-test). Initial mastery of course material was assessed via a 94-item multiple-choice final exam (T1) given in the semester 4 clinical pathology course. Retention of course material from semester 4 to year 4 was assessed via a 55-item multiple-choice pre-test, administered at the start of the clinical pathology rotation in year 4 while learning/mastery during the clinical rotation was assessed via a 55-item multiple-choice post-test, administered at the end of each clinical pathology rotation. In this study, evidence of knowledge retention between semester 4 and year 4 was 55.5%. There is a small increase in the measure of knowledge gain from the beginning to the end of the rotation. As an added benefit, we were able to use identified trends for retention of knowledge within specific subject areas as a mechanism to evaluate the effectiveness of our course and reallocate additional instructional time to topics with poorer retention.

Comparison of Dilution on Eastern Box Turtle (Terrapene carolina carolina) and Marine Toad (Rhinella marinus) Blood Parameters as Measured on a Portable Chemistry Analyzer.

Biochemical testing is an important clinical tool in evaluating the physiology of reptiles and amphibians. Suitable point of care analyzers can allow for rapid delivery of results, but small patient size can inhibit sufficient sample collection. This study evaluated the utility of sample dilution with sterile distilled water as a means of biochemical evaluation when sample volume is limited. Blood was collected from 12 eastern box turtles (Terrapene carolina carolina) and 12 marine toads (Rhinella marinus) and analyzed via i-STAT CHEM8+ cartridges. Two undiluted samples and two samples diluted 1 : 1 with sterile water were evaluated immediately following collection for each animal in the study. Analytes reported in the diluted samples were limited to glucose, ionized calcium, and total carbon dioxide. The expected dilution ratio value of diluted to undiluted samples was 0.5, of which glucose in both turtles and toads was nearest. Dilution ratio values for ionized calcium, however, were higher than expected in both turtles and toads. Sample dilution is not recommended for most analytes included on the CHEM8+ cartridge due to values occurring outside the limits of detection for the analyzer. Glucose and ionized calcium values obtained on diluted samples should be interpreted with caution but may provide clinical utility in reptile and amphibian patients where sample volume is limited.

Hematology of the keeled box turtle (Cuora mouhotii).

A healthy adult, intact female keeled box turtle (Cuora mouhotii) was found to have a marked heterophilic leukocytosis using normal hematologic parameters established for the eastern box turtle (Terrapene carolina carolina), a related chelonian species. This animal was monitored with serial complete blood counts (CBCs) over the next 15 years despite remaining asymptomatic for an infectious condition. Retrospective CBC data were compiled from 38 presumably healthy keeled box turtles to establish hematologic values for comparison in this species. Using this species-specific data, over the 15-year period, the female keeled box turtle had two times where the white blood cell (WBC) count was greater than 2 standard deviations (SD) above the mean, six times where the WBC count was greater than 1 SD above the mean, six times where the PCV was greater than 2 SD above the mean, and eight times where the PCV was greater than 1 SD above the mean. Infection and inflammation are the most common causes of leukocytosis in reptiles; however, given the clinical presentation of this patient, it was postulated that these clinicopathologic changes could be secondary to a stress response. Establishing reference intervals and understanding how stress impacts CBC parameters are important for evaluating the health status of keeled box turtles kept in captivity and for assessing the effects of environmental changes on the health status of wild populations of this endangered chelonian species.

Cancer-testis antigens in canine histiocytic sarcoma and other malignancies.

Cancer-testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies, mostly solid tumours, due to epigenetic de-repression. Normally expressed only in fetal or gametogenic tissues, CTAs are tantalizing immunotherapy targets, since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers, and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this, the ligandome binding the dog leukocyte antigen (DLA)-88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA-derived peptides, which could serve as CD8+ T-cell epitopes. Twenty-two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumour and normal tissue by quantitative and end-point RT-PCR. The ortholog of an established CTA, IGF2BP3, had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis-enhanced proteins were also assessed. AKR1E2, SPECC1 and TPX2 were expressed variably in HS and T-cell lymphoma biopsies, but also at high levels in critical tissues, including kidney, brain and marrow, diminishing their utility. A more tissue-restricted candidate, NT5C1B, was detected in T-cell lymphomas, but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach, proceeding from identification of MHC class I-presented peptides to a comparative RNA expression survey of tumours and normal tissues.

Multiloculated solitary (unicameral) bone cyst in a young dog.

A 20-month-old female spayed Staffordshire Terrier (22.3 kg) presented to the Orthopedic Surgery Service at North Carolina State University Veterinary Teaching Hospital for evaluation of a 6-week history of toe-touching to nonweight-bearing lameness in the right hind limb. Radiographs of the right stifle revealed a multiloculated lytic lesion of the distal femur, with a large open lytic zone centrally, numerous osseous septations peripherally, and focal areas of cortical thinning and loss. An aspirate of the right distal femoral lesion yielded mildly cloudy serosanguineous fluid. Cytologic examination of the fluid revealed a pleomorphic population of discrete cells that exhibited marked anisocytosis and anisokaryosis and a variable nuclear-to-cytoplasmic (N:C) ratio, which were interpreted as probable neoplastic cells, with few macrophages, and evidence of hemorrhage. Given the clinical signs of pain, lesion size, and concern for malignant neoplasia, amputation of the right hind limb was performed. Histologically, the lesion had undulating walls 1-3 mm thick with a continuous outer layer of dense fibrous tissue and an inner layer composed of reactive cancellous bone with no cortical compacta remaining. Remnants of thin fibrous or fibro-osseous septa projected from the bony wall into the cyst lumen. The final histologic diagnosis was a benign multiloculated solitary (unicameral) bone cyst of the distal right femur. Based on the histopathologic findings, it was speculated that the cells identified on cytology were a mixture of developing osteoclasts, osteoblasts, endothelial, and stromal cells. This is the first report describing the cytologic examination of a solitary bone cyst in veterinary medicine.