Atypical granulation in neutrophils of a domestic shorthair cat.

A 13-year-old male domestic short-hair cat presented for evaluation of labored breathing, hyporexia, and lethargy. Pertinent initial diagnostics yielded leukocytosis, characterized by neutrophilia and monocytosis. Numerous small, round, magenta granules were observed within all neutrophils in Wright-Giemsa-stained blood films on the day of presentation and the day thereafter. No other neutrophil morphologic abnormalities were present, making cytoplasmic toxicity highly unlikely. Hyperadrenocorticism was diagnosed based on the lack of suppression in a low-dose dexamethasone suppression test, and without other diagnostics, the cat was discharged on trilostane therapy. Neutrophil granules did not stain with Alcian blue pH 1.0, periodic acid-Schiff (PAS), PAS and Alcian blue pH 2.5, and toluidine blue. Electron microscopy identified no differences in the morphology of the secretory granules or other neutrophil features. Metabolic screening tests of the cat's urine did not identify a genetic metabolic disorder. However, serum α- and ß -hexosaminidase (HexA and HexB) activities were 4.3% and 0% of normal controls, respectively, which is supportive of GM2-gangliosidosis, that is, Sandhoff disorder. However, the historical, clinical, and electron microscopy findings did not provide evidence to confirm this genetic defect. To the author's knowledge, this is the first case of magenta-staining granules within neutrophils in a breed other than a Birman, Siamese, or Himalayan.

Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor.

Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid-onset dementia and death. However, elimination of endogenous PrP halts prion disease progression. In this study, we describe Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM), a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates endogenous DNA methyltransferases, thereby reducing transgene size and cytotoxicity. When delivered to the mouse brain by systemic injection of adeno-associated virus (AAV), Prnp-targeted CHARM ablates PrP expression across the brain. Furthermore, we have temporally limited editor expression by implementing a kinetically tuned self-silencing approach. CHARM potentially represents a broadly applicable strategy to suppress pathogenic proteins, including those implicated in other neurodegenerative diseases.

An AAV capsid reprogrammed to bind human transferrin receptor mediates brain-wide gene delivery.

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an adeno-associated virus (AAV) capsid, BI-hTFR1, that binds human transferrin receptor (TfR1), a protein expressed on the blood-brain barrier. BI-hTFR1 was actively transported across human brain endothelial cells and, relative to AAV9, provided 40 to 50 times greater reporter expression in the CNS of human TFRC knockin mice. The enhanced tropism was CNS-specific and absent in wild-type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared with AAV9. These findings establish BI-hTFR1 as a potential vector for human CNS gene therapy.

Cytochemical staining of leukocytes and platelets in the Florida manatee (Trichechus manatus latirostris): identification of a bilobed monocyte similar to other members of the Paenungulata.

Manatees (Antillean-, Amazonian, and African-) and dugongs belong to the Order Sirenia, and when combined with elephants and rock hyraxes, form the Paenungulata. A bilobed mononuclear cell has previously been identified in elephants and rock hyraxes, but not in manatees and dugongs, with cytochemical staining identifying these cells as bilobed monocytes in elephants. The objective of this study was to characterize leukocytes (white blood cells, WBC) and platelets in blood films of Florida manatees (Trichechus manatus latirostris; n = 8) using one routine hematological (Wright-Giemsa) and eight cytochemical stains: alkaline phosphatase (ALP), α-naphthyl butyrate esterase (ANBE), chloroacetate esterase (CAE), Luna, myeloperoxidase (MPx), periodic acid-Schiff (PAS), Sudan black B (SBB), and toluidine blue (TB). Heterophils and lymphocytes comprised most of the WBC, with low numbers of eosinophils, basophils, and monocytes. Additionally, 1-3% of the WBC were bilobed mononuclear cells. Bilobed mononuclear cell proportions were similar to rock hyraxes, but lower than elephants (approximate range 20-60%). Heterophils and eosinophils were positive for MPx, ALP, SBB, and PAS, with heterophils also being positive for CAE. Most of the lymphocytes were positive for ANBE and they were variably positive for CAE. Monocytes and bilobed mononuclear cells had similar cytochemical staining reactions (variably positive for all stains, except Luna and TB), supporting a monocytic origin, like elephants. Platelets were ANBE- and PAS-positive. Luna stain was useful for identifying eosinophils and TB was uninformative. This study provides new information on the morphological features and cytochemical staining characteristics of WBC and platelets and will aid in obtaining accurate hematological data of Florida manatees.

An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery.

Developing vehicles that efficiently deliver genes throughout the human central nervous system (CNS) will broaden the range of treatable genetic diseases. We engineered an AAV capsid, BI-hTFR1, that binds human Transferrin Receptor (TfR1), a protein expressed on the blood-brain barrier (BBB). BI-hTFR1 was actively transported across a human brain endothelial cell layer and, relative to AAV9, provided 40-50 times greater reporter expression in the CNS of human TFRC knock-in mice. The enhanced tropism was CNS-specific and absent in wild type mice. When used to deliver GBA1, mutations of which cause Gaucher disease and are linked to Parkinson's disease, BI-hTFR1 substantially increased brain and cerebrospinal fluid glucocerebrosidase activity compared to AAV9. These findings establish BI-hTFR1 as a promising vector for human CNS gene therapy.

Left shift and toxic change in heterophils and neutrophils of non-mammalian vertebrates: A comparative review, image atlas, and practical considerations.

Heterophils and neutrophils are important first cellular responders to inflammatory conditions. In addition to quantitative shifts in the numbers of these cells in blood, inflammatory disease states often have accompanying increases in immature precursor stages (left shift) and/or evidence of toxic change on blood film evaluation. Recognition of left shift and toxic change morphologies is a salient diagnostic finding with clinical relevance across species. The objectives of this report are to (a) review heterophil and neutrophil function and structure across the vertebrate animal kingdom, (b) compare morphologic features of left shift and toxic change in heterophils and neutrophils of non-mammalian vertebrates (NMV) to mammals, (c) provide an image guide demonstrating the breadth of morphologic diversity of heterophil and neutrophil lineages in health and disease across taxa, and (d) discuss practical considerations for clinical pathologists and other professionals involved in the recognition and interpretation of observations in the inflammatory leukogram of NMV.

Claisen rearrangements of equilibrating allylic azides.

Equilibrating mixtures of allylic azide-containing allylic alcohols or allylic 2-tolylsulfonylacetic esters undergo Johnson-Claisen or Ireland-Claisen rearrangement reactions to give unsaturated γ-azidoesters and -acids, respectively. Decarboxylation of the acids under basic conditions gives azidosulfones, with moderate to high diastereoselectivity.

Hemolysis, myopathy, and cardiac disease associated with hereditary phosphofructokinase deficiency in two Whippets.

Two male castrated Whippet littermates were presented at 1 year of age for pallor, tachycardia, systolic heart murmur, dark yellow to orange feces, intermittent lethargy, pigmenturia, and muscle shivering or cramping after exercise. Persistent macrocytic hypochromic anemia with marked reticulocytosis and metarubricytosis was found when CBC results were compared with reference values for Whippets. Increased serum creatine kinase activity and hyperkalemia also were sometimes present over the 4-year period of evaluation. Progressively increasing serum concentrations of N-terminal prohormone brain natriuretic peptide suggested cardiac disease. Erythrocytes from the whippets were less osmotically fragile but more alkaline fragile than those from control dogs. Erythrocyte phosphofructokinase (PFK) activities and 2,3-diphosphoglycerate concentrations were decreased. Restriction enzyme-based DNA test screening and DNA sequencing revealed the same mutation in the muscle-PFK gene of the Whippets as seen in English Springer Spaniel dogs with PFK deficiency. This is the first report of PFK deficiency in Whippet dogs. In addition to causing hemolysis and exertional myopathy, heart disease may be a prominent clinical component of PFK deficiency in this breed and has not been previously recognized in PFK-deficient English Springer Spaniels.

B-cell lymphoma with Mott cell differentiation in two young adult dogs.

Two young adult dogs with gastrointestinal signs were each found to have an intra-abdominal mass based on physical examination and diagnostic imaging. On exploratory laparotomy, small intestinal masses and mesenteric lymphadenopathy were found in both dogs; a liver mass was also found in dog 1. Cytologic and histologic examination of intestinal and liver masses and mesenteric lymph nodes revealed 2 distinct lymphoid cell populations: lymphoblasts and atypical Mott cells. With Romanowsky stains, the atypical Mott cells contained many discrete, clear to pale blue cytoplasmic inclusions consistent with Russell bodies that were positive by immunohistochemistry for IgM and CD79a in both dogs and for IgG in dog 2. The Mott cells and occasional lymphoblasts stained strongly positive with periodic acid-Schiff. Using flow cytometric immunophenotyping in dog 1, 60% of peripheral blood mononuclear cells and 85% of cells in an affected lymph node were positive for CD21, CD79a, IgM, and MCH II, indicative of B-cells. With electron microscopy, disorganized and dilated endoplasmic reticulum was seen in Mott cells in tumors from both dogs. Antigen receptor gene rearrangement analysis of lymph node and intestinal masses indicated a clonal B-cell population. Based on cell morphology, tissue involvement, and evidence for clonal B-cell proliferation, we diagnosed neoplasms involving Mott cells. To the authors' knowledge, this is the second report of Mott cell tumors or, more appropriately, B-cell lymphoma with Mott cell differentiation, in dogs. More complete characterization of this neoplasm requires further investigation of additional cases. This lymphoproliferative disease should be considered as a differential diagnosis for canine gastrointestinal tumors.

Hematology of healthy Florida manatees (Trichechus manatus).

BACKGROUND: Hematologic analysis is an important tool in evaluating the general health status of free-ranging manatees and in the diagnosis and monitoring of rehabilitating animals. OBJECTIVES: The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender. METHODS: Blood was collected from 55 free-ranging and 63 captive healthy manatees. Most analytes were measured using a CELL-DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations. RESULTS: Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free-ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals. CONCLUSIONS: Hematologic findings were generally similar between captive and free-ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals.

Comparison of blood aminotransferase methods for assessment of myopathy and hepatopathy in Florida manatees (Trichechus manatus latirostris).

Muscle injury is common in Florida manatees (Trichechus manatus latirostris). Plasma aspartate aminotransferase (AST) is frequently used to assess muscular damage in capture myopathy and traumatic injury. Therefore, accurate measurement of AST and alanine aminotransferase (ALT) is important in managed, free-ranging animals, as well as in those rehabilitating from injury. Activities of these enzymes, however, are usually not increased in manatees with either acute or chronic muscle damage, despite marked increases in plasma creatine kinase activity. It is hypothesized that this absence of response is due to apoenzymes in the blood not detected by commonly used veterinary assays. Addition of coenzyme pyridoxal-5-phosphate (P5P or vitamin B6) should, therefore, result in higher measured enzyme activities. The objective of this study was to determine the most accurate, precise, and diagnostically useful method for aminotransferase measurement in manatees that can be used in veterinary practices and diagnostic laboratories. Additionally, appropriate collection and storage techniques were assessed. The use of an optimized commercial wet chemical assay with 100 micromol P5P resulted in a positive bias of measured enzyme activities in a healthy population of animals. However, AST and ALT were still much lower than that typically observed in domestic animals and should not be used alone in the assessment of capture myopathy and muscular trauma. Additionally, the dry chemistry analyzer, typically used in clinics, reported significantly higher and less precise AST and ALT activities with poor correlation to those measured with wet chemical methods found in diagnostic laboratories. Therefore, these results cannot be clinically compared. Overall, the optimized wet chemical method was the most precise and diagnostically useful measurement of aminotransferase in samples. Additionally, there was a statistically significant difference between paired serum and plasma measurement, indicating that separate reference intervals should be established for serum and plasma. Finally, storage of these enzymes at -70 degrees C for 1 mo resulted in up to a 25% decrease in enzymatic activity in manatee plasma.

Laboratory findings in acute Cytauxzoon felis infection in cougars (Puma concolor couguar) in Florida.

Intraerythrocytic piroplasms, morphologically indistinguishable from Cytauxzoon felis, were identified in stained blood films from more than one third of free-ranging cougars (Puma concolor couguar) in southern Florida in a study that failed to demonstrate negative effects of piroplasm infection on measured hematologic parameters. However, a recent study with a nested 18s rRNA polymerase chain reaction (PCR) assay identified only 9% of the free-ranging cougars in southern Florida as infected with C. felis but found 83% of these animals were infected with an unnamed small Babesia sp. In this study, hematology and clinical chemistry parameters were determined during the initial appearance of piroplasms in stained blood films of three western cougars housed in northern Florida. One animal became ill, but the remaining two animals did not exhibit clinical signs of disease. The hematocrit decreased in all three cougars concomitant with the first recognized parasitemia. A regenerative response to anemia (increased polychromasia, increased mean cell volume, and increased red cell distribution width) was recognized in two cougars that were examined twice during the following 2 weeks. Thrombocytopenia and probable leukopenia occurred in one animal. The most consistent clinical chemistry findings were increased serum bilirubin concentrations and increased alanine aminotransferase and aspartate aminotransferase activities at the time of initial recognition of parasitemia. Serum protein findings were not consistent in these cougars. The use of PCR and determination of 18S rRNA gene sequences in the blood from these three animals revealed infection with C. felis, but not with the Babesia sp. In this report, we demonstrate that mild hemolytic anemia, and probably liver injury, occurs concomitant with the initial discovery of C. felis piroplasms in stained blood films.

Clinical biochemistry in healthy manatees (Trichechus manatus latirostris).

Florida manatees (Trichechus manatus latirostris) are endangered aquatic mammals living in coastal and riverine waterways of Florida and adjacent states. Serum or plasma biochemical analyses are important tools in evaluating the health of free-ranging and captive manatees. The purpose of this study was to measure diagnostically important analytes in the plasma of healthy manatees and to determine whether there was significant variation with respect to location (free-ranging versus captive), age class (small calves, large calves, subadults, adults), and gender. No significant differences in plasma sodium, potassium, bilirubin, glucose, alanine aminotransferase, or creatine kinase were found among these classes of animals. Compared to free-ranging manatees, captive animals had significantly lower mean concentrations of plasma chloride, phosphate, magnesium, triglycerides, anion gap, and lactate. Captive manatees had significantly higher mean values of total CO2, calcium, urea, creatinine, alkaline phosphatase, gamma-glutamyltransferase, total protein, albumin, and albumin/globulin ratio than did free-ranging animals. Differences in the environments of these two groups, including diet, temperature, salinity, and stress, might account for some of these results. The higher plasma lactate and anion gap concentrations and lower total CO2 concentrations of free-ranging manatees were probably due to greater exertion during capture, but the lack of elevated plasma creatine kinase activity relative to captive animals indicates that there was no serious muscle injury associated with capture. Plasma phosphate decreased and total globulins increased with age. Plasma cholesterol and triglyceride concentrations were highest in small calves. Plasma aspartate aminotransferase was higher in large calves than in adults and subadults, and the albumin/ globulin ratio was higher in subadults than in adults. Plasma total CO2 was higher and chloride was slightly lower in females than in males.

The feline blood film.

Practical relevance: Many veterinary practices have invested in quality automated hematology instruments for use in-house. However, regardless of their specific choice of analyzer, there are important hematology findings that can only be determined by microscopic examination of stained blood films. For this reason, and also for the purpose of quality control for the analyzer, a quick blood film review should be performed alongside every automated complete blood count. Even those practices that submit their blood samples to outside diagnostic laboratories for evaluation, still require the capability to examine stained blood films in emergency situations. Series outline: This is the second of a two-part article series that aims to familiarize the practitioner with normal findings on feline blood films, with a particular focus on unique features in the cat, as well as to assist with interpretation of common abnormalities. Part 2 focuses on the morphology of feline leukocytes and platelets in health and disease. Evidence base: The information and guidance offered is based on the published literature and the author's own extensive clinical pathology research.

The feline blood film.

Practical relevance: Many veterinary practices have invested in quality automated hematology instruments for use in-house. However, regardless of the specific choice of analyzer, there are important hematology findings that can only be determined by microscopic examination of stained blood films. For this reason, and also for the purpose of quality control for the analyzer, a quick blood film review should be performed alongside every automated complete blood count. Even those practices that submit their blood samples to outside diagnostic laboratories for evaluation, still require the capability to examine stained blood films in emergency situations. Series outline: This is the first of a two-part article series that aims to familiarize the practitioner with normal findings on feline blood films, with a particular focus on unique features in the cat, as well as to assist with interpretation of common abnormalities. Part 1 focuses on how to prepare and examine blood films in order to maximize the reliability of the information they convey, and describes the morphology of feline erythrocytes in health and disease. Evidence base: The information and guidance offered is based on the published literature and the author's own extensive clinical pathology research.

Bone Marrow Aspirate Evaluation.

Important steps in bone marrow aspirate evaluation include determining if bone marrow evaluation is indicated; using appropriate aspirate collection, smear preparation, and staining techniques; and performing a systematic approach for the cytologic evaluation. The cytologic evaluation of bone marrow requires knowledge of the morphology of bone marrow cell types, the proportion of these cell types normally present, and the ability to evaluate overall cellularity of bone marrow. Accurate interpretation of bone marrow cytologic findings depends on evaluation of a current complete blood cell count. These components are the pillars of getting the most useful information in the diagnosis of hematologic disorders.

Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses.

Deficiencies of enzymes involved in erythrocyte metabolism can have significant effects on erythrocyte function and survival. Animals with pyruvate kinase (PK) or phosphofructokinase (PFK) deficiencies have shortened erythrocyte life spans and regenerative anemia. PK-deficient dogs (but not PK-deficient cats) develop progressive myelofibrosis and osteosclerosis of bone marrow and hemochromatosis and cirrhosis of the liver. PFK-deficient dogs have sporadic episodes of hyperventilation-induced intravascular hemolysis and hemoglobinuria. Cytochrome b5 reductase (Cb5R) deficiency in dogs and cats results in persistent methemoglobinemia and cyanotic mucous membranes. Severe deficiency of glucose-6-phosphate dehydrogenase, the rate-controlling enzyme in the pentose phosphate pathway, resulted in anemia with eccentrocytosis in an American saddlebred colt. Horses with erythrocyte flavin adenine dinucleotide (FAD) deficiency have both eccentrocytosis (attributable to severe deficiency in glutathione reductase activity) and methemoglobinemia (attributable to Cb5R deficiency); the dual enzyme deficiency occurs because FAD is a required cofactor for both enzymes. Erythrocyte enzyme deficiencies do not usually shorten life expectancy, except for PK-deficient dogs and potentially PFK-deficient dogs during a hemolytic crisis. Although enzyme deficiencies are rare causes of anemia and methemoglobinemia, the ability to diagnose deficient animals allows for the possibility of eliminating these undesirable traits in future breeding. DNA-based assays are available for PK and PFK deficiencies; whereas, biochemical tests of enzyme activity are required for other deficiencies. Continued research is needed to document additional enzyme deficiencies that likely occur and to develop additional DNA-based assays to detect heterozygous animals.