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Anemia is a clinically important syndrome in small ruminants. Anemia can be divided into regenerative and nonregenerative forms. Differentials for regenerative anemia include hemorrhage owing to gastrointestinal or external parasitism or hemostatic disorders, and hemolysis owing to infectious, osmotic, toxic, and nutritional causes. Differentials for nonregenerative anemia include inflammatory and chronic diseases, renal failure, pancytopenia, copper deficiency, and heavy metal toxicosis. Iron deficiency anemia can be caused by chronic gastrointestinal and external hemorrhage or nutritional deficiency and may be mildly regenerative or nonregenerative. Appropriate diagnostic tests are described along with treatments, including blood transfusion, parasite control, and prevention.

Background Selenium or alpha‐tocopherol deficiency can cause neuromuscular disease. Beta‐carotene has limited documentation in horses. Objective To evaluate the effect of owner practices on plasma beta‐carotene concentration and risk of selenium and alpha‐tocopherol deficiencies. Animals Three‐hundred and forty‐nine adult (≥1 year), university and privately owned horses and mules. Methods Cross‐sectional study. Whole blood selenium, plasma alpha‐tocopherol, and plasma beta‐carotene concentrations were measured once. Estimates of daily selenium and vitamin E intake, pasture access, and exercise load were determined by owner questionnaire. Data were analyzed using t tests, Mann‐Whitney tests, parametric or nonparametric analysis of variance (ANOVA), Kruskal‐Wallis test, Spearman's correlation and contingency tables (P 6 h/d) and daily provision of ≥500 IU of vitamin E was associated (P

Since the 1980s society has been largely satisfied with modern manufacturing’s production practice. We are able to develop supply chains that can produce today’s highly complex and advanced products and are able to scale mass production to meet much of society’s demands. However, our growing reliance on a constrained set of production practices leaves society exposed when societal conditions drastically change (e.g., COVID-19). Mass-customisation also suggests that the future manufacturing landscape will be far more volatile requiring a response that cannot be achieved today. Sustainable manufacturing is also of prominent and critical importance, and a reliance on other nation’s production capabilities has concerned many nations potentially leaving them exposed during trade negotiations and diplomacy. While concerns exist, the rapidly maturing field of Additive Manufacturing (AM) and its highly distributed and diverse nature may be able to alleviate them. If we can broker it effectively, AM can come together as one to tackle local, regional, national, and international needs. In this paper, we take a closer look at the drivers that are requiring us to re-think production practice. This is followed by a proposition that effective brokering of AM could mitigate the drivers. The paper then summarises the work the manufacturing community needs to perform in order to make it a reality. © 2022, The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd.

Background Renal lymphoma in dogs is rare and has a poor prognosis. Granular lymphocyte morphology is rarely reported in canine renal lymphoma. Mild to moderate polycythemia is reported in a number of canine renal lymphoma cases. Case presentation A 10-year-old Labrador retriever presented to a university veterinary teaching hospital after a 1-month history of polyuria, polydipsia, and pollakiuria and a 2-week history of abdominal distention, lethargy, and increased respiratory effort. Abdominal ultrasound showed a wedge-shaped to rounded, heterogeneously hypoechoic mass lesion in the left kidney. Cytologic analysis of a percutaneous aspirate of the mass was consistent with lymphoma of granular lymphocytes. Severe polycythemia (hematocrit 0.871) was noted on a complete blood cell count. Clonality analysis identified a clonally rearranged T-cell receptor (TCR) gene and immunohistochemical staining was CD3+, CD79a- and CD11d+, supporting cytotoxic T-cell lymphoma. Conclusions To our knowledge, this is the first report of renal cytotoxic T-cell lymphoma with severe polycythemia in a dog. Severe polycythemia and renal cytotoxic T-cell lymphoma are both rare in dogs; this report adds to the body of knowledge on these conditions.

Teaching approaches to veterinary clinical pathology in the final (clinical) year of veterinary school are often different than those for other specialties. Anecdotally, many schools teach these rotations separately from the routine diagnostic service, but minimal published data are available on this topic or on approaches to teaching and assessment in these rotations. An online survey of 69 veterinary institutions around the world was conducted in 2019. A total of 30 completed surveys were received from 10 countries; 22 completed responses were from North American institutions (73.3%). Survey question categories included information on basic rotations, including microscopy format, personnel involved in instruction, and assessment methods; information on advanced rotations; and challenges and successes with clinical pathology instruction. Data were analyzed and, when appropriate, compared with results from a similar survey conducted in 1997. Formats and content varied greatly among institutions. Several shifts in teaching strategies and rotation format over time were found since the 1997 survey, including increased use of projection microscopy and decreased use of multiheaded microscopy in 2019. More teaching by medical technologists and residents, less teaching by faculty, and a significant increase in the number of students per rotation were seen in 2019 compared with 1997. Several free-text comments referred to challenges related to increasing class size. These data and the comparison with the prior survey highlight common challenges and potential solutions to final-year clinical pathology instruction. Creation of specific, measurable objectives for clinical pathology competence may aid future development and refinement of clinical pathology teaching.

Systemic immune responses in cancer patients are of tremendous importance, both to advance understanding of disease mechanisms and for development of new diagnostic testing. Minimal published information is available on the systemic cytokine response in canine osteosarcoma (OS) patients. The goal of this study was to investigate serum cytokine alterations present in OS patients at the time of diagnosis. Serum samples from 22 canine OS patients at the time of diagnosis and 18 healthy control dogs were evaluated via multiplex immunoassay for 14 analytes. Significant increases in serum interleukin-8 (IL-8) and interleukin-12p40 (IL-12p40) concentrations were found in OS patients when compared to healthy controls. The results correlate with several published studies on serum cytokine alterations in human OS patients. These data add to the growing body of knowledge on immunologic alterations in OS, including potential immunomodulatory therapy of canine patients, and support future studies on serum cytokine testing to investigate diagnostic and prognostic utility.

A significant portion of lung development is completed postnatally during alveolarization, rendering the immature lung vulnerable to inflammatory stimuli that can disrupt lung structure and function. Although the NF-κB pathway has well-recognized pro-inflammatory functions, novel anti-inflammatory and developmental roles for NF-κB have recently been described. Thus, to determine how NF-κB modulates alveolarization during inflammation, we exposed postnatal day 6 mice to vehicle (PBS), systemic lipopolysaccharide (LPS), or the combination of LPS and the global NF-κB pathway inhibitor BAY 11-7082 (LPS + BAY). LPS impaired alveolarization, decreased lung cell proliferation, and reduced epithelial growth factor expression. BAY exaggerated these detrimental effects of LPS, further suppressing proliferation and disrupting pulmonary angiogenesis, an essential component of alveolarization. The more severe pathology induced by LPS + BAY was associated with marked increases in lung and plasma levels of macrophage inflammatory protein-2 (MIP-2). Experiments using primary neonatal pulmonary endothelial cells (PEC) demonstrated that MIP-2 directly impaired neonatal PEC migration in vitro; and neutralization of MIP-2 in vivo preserved lung cell proliferation and pulmonary angiogenesis and prevented the more severe alveolar disruption induced by the combined treatment of LPS + BAY. Taken together, these studies demonstrate a key anti-inflammatory function of the NF-κB pathway in the early alveolar lung that functions to mitigate the detrimental effects of inflammation on pulmonary angiogenesis and alveolarization. Furthermore, these data suggest that neutralization of MIP-2 may represent a novel therapeutic target that could be beneficial in preserving lung growth in premature infants exposed to inflammatory stress.

Acute inflammatory processes can trigger increased production of acute phase proteins (APPs) that can be useful biomarkers of inflammation. APPs are diverse and include proteins involved in coagulation, opsonization, iron regulation, and limitation of tissue injury. Haptoglobin, serum amyloid A, and alpha-1 acid glycoprotein have been proposed as useful APPs in goats. APPs can differ markedly by species, therefore species-specific reference intervals and studies are necessary. The objective of this study was to determine species-specific reference intervals for 4 APPs in goats. Haptoglobin, serum amyloid A, lipopolysaccharide binding protein, and alpha-1 acid glycoprotein were measured in in 54 clinically normal adult goats. APPs were measured using goat-specific commercial enzyme-linked immunosorbent assay kits. Results were analyzed by 1-way analysis of variance to compare sexes and breeding status. Reference Value Advisor was used to calculate reference limits according to the IFCC-CLSI guidelines. Only 1 APP was found to vary in healthy animals; serum haptoglobin was increased in lactating animals and decreased in pregnant does in their second trimester when compared with open, nonlactating does. No sex-based differences were seen for any of the APPs measured. We report normal reference intervals for 4 serum APPs that may be useful as disease markers. Haptoglobin should be interpreted with caution in animals with unknown pregnancy status. Further studies are needed to determine whether these APPs are useful biomarkers in goat disease states.

The role of host type I IFN signaling and its interaction with other immune pathways during bacterial infections is incompletely understood. Type II IFN signaling plays a key role during numerous bacterial infections including granulocytic anaplasmosis (GA) caused by Anaplasma phagocytophilum infection. The function of combined type I and type II IFN signaling and their potential synergism during GA and similar tick-borne diseases is a topic of current research investigation. The goal of this study was to evaluate 2 mouse models of absent type I/type II IFN signaling in experimental A. phagocytophilum infection to determine the effects of background strain. Mice lacking both type I and type II IFN receptor signaling (IFNAR−/−/IFNGR–/–) on either the 129/SvEv or C57BL/6J genetic background were evaluated at days 0, 6, 8, and 12 of infection. Pathogen burden in multiple organs was largely similar between strains of infected mice, with few significant differences. Background strain influenced the immune response to infection. Mice of the 129/SvEv strain developed more severe hematologic abnormalities, particularly more severe leukocytosis with marked neutrophilia and lymphocytosis, throughout acute infection. Histopathologic changes occurred in infected mice of both strains and varied in severity by organ. 129/SvEv mice developed more severe pathologic changes in spleen and bone marrow, whereas C57BL/6J mice developed more severe renal pathology. This work highlights the importance of mouse background strain in dictating pathophysiologic response to infection and informs future work regarding the loss of type I and type II IFN signaling on the immune response during GA.

The chytrid fungus, Batrachochytrium dendrobatidis (Bd), has been identified as a major cause of the recent worldwide amphibian decline. Numerous species in North America alone are under threat or have succumbed to Bd-driven population extinctions. The American bullfrog (Lithobates catesbeianus) has been reported as a tolerant carrier of Bd. In this report, we used a qPCR assay to test 120 archived American bullfrog specimens collected between 1924 and 2007 in California, USA and Baja California, Mexico. The overall prevalence of Bd infection in this archived population of L. catesbeianus was 19.2%. The earliest positive specimen was collected in Sacramento County, California, USA in 1928 and is to date the earliest positive archived Bd specimen reported globally. These data demonstrate that Bd-infected wild amphibians have been present in California longer than previously known.

Background Clinical pathology of rodents is hindered by sample volume limitations. A single whole heparinized blood sample is often submitted for hematologic and clinical chemistry analysis in exploratory research settings, and sample dilution may be required. Published information on the potential impact of sample dilution and heparin use on hematology variables in rodents is sparse. Objectives The purpose of the study was to evaluate the effects of sample dilution and of anticoagulant on hematologic analysis of mouse and rat blood samples on the Sysmex XT-2000iV. Methods Mice and rats were obtained from various ends of study research projects, and whole blood was collected via terminal cardiocentesis in lithium heparin, and additionally in EDTA when paired samples were obtained from rats. Hematology analytes were measured on the Sysmex XT-2000iV straight and diluted from ×2 to ×5. Results Significant differences between heparinized samples analyzed straight vs diluted were found for MCV and MCHC, with a bias for several additional variables. Significant differences between paired heparinized and EDTA-anticoagulated samples at each dilution point were found for most variables, with the largest differences found in platelet count. Evidence of platelet clumping presumably due to heparin exposure was noted in numerous samples. Conclusions Dilution-induced changes occur in some hematologic variables and may render dilution unacceptable in the exploratory research environment. Many variables, most notably platelet count, differ based on the anticoagulant used, and values from heparinized vs EDTA-anticoagulated samples should not be directly compared.

Anaplasma phagocytophilum is a tick-borne rickettsial pathogen that provokes an acute inflammatory response during mammalian infection. The illness caused by A. phagocytophilum , human granulocytic anaplasmosis, occurs irrespective of pathogen load and results instead from host-derived immunopathology. Thus, characterizing A. phagocytophilum genes that affect the inflammatory process is critical for understanding disease etiology. By using an A. phagocytophilum Himar1 transposon mutant library, we showed that a single transposon insertion into the A. phagocytophilum dihydrolipoamide dehydrogenase 1 gene ( lpda1 [ APH _ 0065 ]) affects inflammation during infection. A. phagocytophilum lacking lpda1 revealed enlargement of the spleen, increased splenic extramedullary hematopoiesis, and altered clinicopathological abnormalities during mammalian colonization. Furthermore, LPDA1-derived immunopathology was independent of neutrophil infection and correlated with enhanced reactive oxygen species from NADPH oxidase and nuclear factor (NF)-κB signaling in macrophages. Taken together, these findings suggest the presence of different signaling pathways in neutrophils and macrophages during A. phagocytophilum invasion and highlight the importance of LPDA1 as an immunopathological molecule.

Background The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. Objectives The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. Methods Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. Results For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 107 and after processing was significantly higher at 7.8 ± 3.3 × 107 (P

Objective—To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. Animals—13 healthy adult Thoroughbreds. Procedures—Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4°C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. Results—Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 μg of streptavidin-PE/1 × 106 RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. Conclusions and Clinical Relevance—Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.

Anaplasma phagocytophilum is an obligate intracellular bacterium that has evolved mechanisms to hijack polymorphonuclear neutrophil (PMN) receptors and signaling pathways to bind, infect, and multiply within the host cell. E-selectin is upregulated during inflammation and is a requisite endothelial receptor that supports PMN capture, rolling, and activation of integrin-mediated arrest. Ligands expressed by PMN that mediate binding to endothelium via E-selectin include sialyl Lewis x (sLex)-expressing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1) and other glycolipids and glycoproteins. As A. phagocytophilum is capable of binding to sLex-expressing ligands expressed on PMN, we hypothesized that acute bacterial adhesion to PMN would subsequently attenuate PMN recruitment during inflammation. We assessed the dynamics of PMN recruitment and migration under shear flow in the presence of a wild-type strain of A. phagocytophilum and compared it with a strain of bacteria that binds to PMN independent of PSGL-1. Acute bacterial engagement with PMN resulted in transient PMN arrest and minimal PMN polarization. Although the wild-type pathogen also signaled activation of β2 integrins and elicited a mild intracellular calcium flux, downstream signals including PMN transmigration and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were inhibited. The mutant strain bound less well to PMN and failed to activate β2 integrins and induce a calcium flux but did result in decreased PMN arrest and polarization that may have been partially mediated by a suppression of p38 MAPK activation. This model suggests that A. phagocytophilum binding to PMN under shear flow during recruitment to inflamed endothelium interferes with normal tethering via E-selectin and navigational signaling of transendothelial migration.

Infection withAnaplasma phagocytophilum, a gram-negative, lipopolysaccharide (LPS)-negative, obligate intracellular bacterium, results in multiple peripheral blood cytopenias. We hypothesized that infection with this organism would result in decreased bone marrow (BM) function and shifts in hematopoietic progenitor cells (HPCs) and lineage-committed cells in a well-established murine model of infection. HPCs and lineage-committed progenitors were enumerated in the BM and spleen during acute infection. BM cytokine production and BM CXCL12 expression were determined. Infection resulted in peripheral blood bicytopenia, marked decreases in the number of lineage-committed HPCs in the BM along with concurrent increases in the number of lineage-committed HPCs in the spleen, and a mixed, predominantly myelosuppressive BM cytokine environment. There was significant downregulation of CXCL12 in BM cells that may have been partially responsible for changes in HPC trafficking observed. Changes occurred in the absence of direct pathogen infection of BM cells. Hematopoietic lineage assessment demonstrated that there was loss of erythrocytes and B lymphocytes from the BM along with increased granulopoiesis. These changes were accompanied by splenomegaly due to lymphoid hyperplasia and increased hematopoiesis, most notably erythropoiesis. These changes largely mimic well-described inflammation and endotoxin-mediated effects on the BM and spleen; however, the numbers of peripheral blood neutrophils appear to be independently modulated as granulocytic hyperplasia does not result in neutrophilia. Our findings highlight a well-conserved series of events that we demonstrate can be instigated by an LPS-negative pathogen in the absence of an endotoxin-mediated acute proinflammatory response.

Hematology is an essential component of veterinary practice. The interpretation of avian blood cells provides many challenges. Practitioners must be able to recognize normal morphology and function of cells to interpret changes in those cells. This article describes the normal morphology of avian erythrocytes, leukocytes, and thrombocytes. Changes observed in erythrocytes and leukocytes during disease and major differential diagnoses are discussed. A brief overview of avian blood parasites is also presented.

Objective—To develop a reticulocyte classification scheme, optimize an avian reticulocyte staining protocol, and compare the percentages of reticulocyte types with polychromatophil percentage in blood samples from birds. Sample Population—Blood samples from a red-tailed hawk and 31 ill birds. Procedures—A single blood sample obtained from a red-tailed hawk (Buteo jamaicensis) was used to optimize the staining protocol. For optimization of the staining protocol, 4 dilutions of whole blood with new methylene blue stain and 4 incubation times were evaluated. From samples submitted for avian CBCs, EDTA-anticoagulated whole blood samples from 31 ill birds were randomly selected and examined to compare polychromatophil and reticulocyte percentages. Reticulocyte staining was performed in all samples by use of a 1:3 (whole blood to new methylene blue) dilution with incubation for 10 minutes at room temperature (approx 22°C); reticulocytes were assessed as a percentage of 1,000 RBCs by 2 independent observers. In Wright-Giemsa–stained blood smears, a polychromatophil percentage was similarly determined. Results—4 avian reticulocyte types were defined: ring-form reticulocytes, aggregate reticulocytes, and 2 subcategories of punctate reticulocytes. A reticulocyte-staining protocol was optimized. Interobserver and intraobserver variations in assessment of reticulocyte and polychromatophil percentages were not significant. A strong positive correlation (Spearman coefficient of rank correlation [ρ] = 0.978) was identified between the percentage of polychromatophils and the percentage of ring-form reticulocytes. Conclusions and Clinical Relevance—Results indicated that quantification of ring-form reticulocytes provides an accurate assessment of erythrocyte regenerative capacity in birds.

Xenopus laevis, the African clawed frog, is commonly used in developmental and toxicology research studies. Little information is available on aged X. laevis; however, with the complete mapping of the genome and the availability of transgenic animal models, the number of aged animals in research colonies is increasing. The goals of this study were to obtain biochemical and hematologic parameters to establish reference intervals for aged X. laevis and to compare results with those from young adult X. laevis. Blood samples were collected from laboratory reared, female frogs (n = 52) between the ages of 10 and 14 y. Reference intervals were generated for 30 biochemistry analytes and full hematologic analysis; these data were compared with prior results for young X. laevis from the same vendor. Parameters that were significantly higher in aged compared with young frogs included calcium, calcium:phosphorus ratio, total protein, albumin, HDL, amylase, potassium, CO2, and uric acid. Parameters found to be significantly lower in aged frogs included glucose, AST, ALT, cholesterol, BUN, BUN:creatinine ratio, phosphorus, triglycerides, LDL, lipase, sodium, chloride, sodium:potassium ratio, and anion gap. Hematology data did not differ between young and old frogs. These findings indicate that chemistry reference intervals for young X. laevis may be inappropriate for use with aged frogs.

A 9-year-old male castrated Scottish terrier was referred to the Radiation Oncology Service at the William R. Pritchard Veterinary Medical Teaching Hospital for palliative radiation therapy of an incompletely excised, recurrent subcutaneous mast cell tumor (MCT) located over the right scapula, and surgical removal of a perianal MCT. Three weeks after initial presentation and prior to the fifth radiation treatment, the patient was presented with cloudiness of the left eye of 3-7 days duration. Ophthalmic consultation revealed 3+ aqueous flare with a dependent, swirling component filling approximately one-third of the anterior chamber. Aqueocentesis was performed under general anesthesia. Cytology revealed mast cells with highly atypical morphology and considered most consistent with neoplasia. The patient died 7 months after pathologic diagnosis of MCT on the right shoulder and 2 months after the cytologic diagnosis of malignant mast cells in the left anterior chamber. To the authors' knowledge, this is the first report of intraocular involvement in a mammal with MCTs, described here as intraocular mastocytosis.

Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism Salmonella enterica serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened TH1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (Tregs), fewer T-bet+ (TH1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened TH1 response with fewer T-bet+ T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet+ TH1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the TH1 immune response in the spleens of mice that are colonized by high levels of S. Typhimurium in the gastrointestinal tract., Author Summary Bacteria belonging to the genus Salmonella are capable of causing long-term chronic systemic infections in specific hosts where they are shed in the feces. These persistently infected individuals include typhoid carriers and they serve as a reservoir for disease transmission. Despite the importance of Salmonella as a human pathogen, relatively little is known about the host immune response to persistent bacterial infections in the context of transmission. We had shown previously in a mouse model of Salmonella infection that mice shedding high levels of Salmonella (>108 bacteria per gram of feces), known as super-shedders, transmit disease to naïve mice. We show here that these super-shedder mice have a unique immune state compared to mice that have lower levels of Salmonella in their gut. The super-shedder immune state is characterized by an active inflammatory immune response with elevated serum IL-6 and high levels of neutrophils in both the gastrointestinal tract and the systemic sites but a dampened adaptive CD4 T helper type1 (TH1) cell response specific to the spleen. Importantly, we show that the blunted adaptive response, as characterized by reduced TH1 cell frequencies and ability to respond to IL-2 and IL-6, is intimately linked to the levels of neutrophils present in the spleen. We go on to show the functional consequences of dampened cytokine responsiveness, as TH1 cells from moderate-shedders are unable to undergo IL-2-mediated expansion when neutrophilia is induced. Additionally, we show that neutrophil control of IL-2 mediated expansion of TH1 cells is independent of infection. In summary, we describe an immune phenotype associated with transmission of a pathogen and a single immune cell type, neutrophils, which control specific aspects of the super-shedder immune state.

Extramedullary hematopoiesis (EMH) is the formation and development of blood cells outside the medullary spaces of the bone marrow. Although widely considered an epiphenomenon, secondary to underlying primary disease and lacking serious clinical or diagnostic implications, the presence of EMH is far from incidental on a molecular basis; rather, it reflects a well-choreographed suite of changes involving stem cells and their microenvironment (the stem cell niche). The goals of this review are to reconsider the molecular basis of EMH based on current knowledge of stem cell niches and to examine its role in the pathophysiologic mechanisms of EMH in animals. The ability of blood cells to home, proliferate, and mature in extramedullary tissues of adult animals reflects embryonic patterns of hematopoiesis and establishment or reactivation of a stem cell niche. This involves pathophysiologic alterations in hematopoietic stem cells, extracellular matrix, stromal cells, and local and systemic chemokines. Four major theories involving changes in stem cells and/or their microenvironment can explain the development of most occurrences of EMH: (1) severe bone marrow failure; (2) myelostimulation; (3) tissue inflammation, injury, and repair; and (4) abnormal chemokine production. EMH has also been reported within many types of neoplasms. Understanding the concepts and factors involved in stem cell niches enhances our understanding of the occurrence of EMH in animals and its relationship to underlying disease. In turn, a better understanding of the prevalence and distribution of EMH in animals and its molecular basis could further inform our understanding of the hematopoietic stem cell niche.

Background Post-transfusion survival of allogeneic RBCs has been reported to be much shorter in horses than in other species. We hypothesized that post-transfusion survival of biotinylated allogeneic equine RBCs would be greater than the survival previously reported from studies using radioactive RBC-labeling techniques. Objective The study objective was to determine post-transfusion survival of N-hydroxysuccinimide (NHS)-biotin-labeled allogeneic equine RBCs transfused into adult horses. Methods Horses were adults and included 5 donors and 5 recipients. All horses were blood-typed, and donors were paired with recipients based upon blood type and crossmatch results. Donor blood was collected in a volume of 4 L into citrate phosphate dextrose adenine-1 and stored for 24 hours, labeled with NHS-biotin, and re-infused into recipients. Post-transfusion blood samples were collected at 15 minutes and at 1, 2, 3, 5, 7, 14, 21, 28, and 35 days. Biotin-labeled RBCs were detected by flow cytometry using streptavidin–phycoerythrin. Post-transfusion survival at 24 hours, lifespan, and half-life of biotinylated RBCs were determined. Results Mean ± SD survival of biotinylated RBCs at 24 hours post-transfusion was 95 ± 24%; the mean lifespan of transfused allogeneic RBCs was 39 days based on calculation of a linear regression survival curve, and mean post-transfusion RBC half-life was 20 days. Conclusions Post-transfusion survival of 24-hour stored equine allogeneic RBCs was greater than previously reported but less than that observed for other companion animal species. Mechanisms for the relatively short post-transfusion lifespan of allogeneic equine RBCs remain unknown and warrant further study.

Infection with a variety of bacterial pathogens results in hematopoietic stem and progenitor cell (HSPC) mobilization. The mechanism and kinetics of HSPC mobilization during infection are largely unknown. Previously, we found altered HSPC activity in bone marrow, spleen and blood during infection with Anaplasma phagocytophilum, the agent of granulocytic anaplasmosis. We hypothesized that altered CXCL12/CXCR4 signaling, a central pathway for HSPC homing to, and retention within, the bone marrow, plays a role in infection-induced alterations in HSPC number and trafficking. Mice were infected with A. phagocytophilum. Lineage-cKit+ HSPCs were enumerated and proliferation determined. CXCL12 and CXCR4 mRNA were quantified along with CXCL12 protein, and CXCR4 surface, intracellular and total protein expression in HSPCs was determined. Increased bone marrow proliferation of HSPCs began at 2 d post-infection followed by HSPC mobilization and splenic homing. Proliferation of resident HSPCs contributed to increased splenic HSPC numbers. Bone marrow CXCL12 mRNA and protein levels were decreased at 4–8 d post-infection concurrent with HSPC mobilization. CXCR4 protein parameters were decreased in bone marrow HSPCs throughout 2–6 d post-infection. Reduction of CXCL12/CXCR4 signaling simultaneously occurs with HSPC mobilization from bone marrow. Findings suggest that deranged CXCL12/CXCR4 signaling plays a causal role in HSPC mobilization during acute A. phagocytophilum infection.

Cathelicidins are small cationic antimicrobial peptides found in many species including primates, mammals, marsupials, birds and even more primitive vertebrates, such as the hagfish. Some animals encode multiple cathelicidins in their genome, whereas others have only one. This report identifies and characterizes feline cathelicidin (feCath) as the sole cathelicidin in domestic cats (Felis catus). Expression of feCath is predominantly found in the bone marrow, with lower levels of expression in the gastrointestinal tract and skin. By immunocytochemistry, feCath localizes to the cytoplasm of neutrophils in feline peripheral blood. Structurally, the mature feCath sequence is most similar to a subgroup of cathelicidins that form linear α-helices. feCath possesses antimicrobial activity against E. coli D31, Salmonella enterica serovar Typhimurium (IR715), Listeria monocytogenes and Staphylococcus pseudintermedius (clinical isolate) similar to that of the human ortholog, LL-37. In contrast, feCath lacks the DNA binding activity seen with LL-37. Given its similarity in sequence, structure, tissue expression, and antimicrobial activity, the cathelicidin encoded by cats, feCath, belongs to the subgroup of linear cathelicidins found not only in humans, but also non-human primates, dogs, mice, and rats.

Peripheral blood cytopenias result from a wide variety of infectious diseases and may significantly complicate the prognosis and outcome of those diseases. Infection with Gram-negative, LPS-negative members of the Anaplasmataceae family, including Anaplasma phagocytophilum and Ehrlichia muris, results in bi- or pancytopenia. The specific mechanisms responsible for the cytopenias are unknown. Viruses and bacteria have profound yet often discrepant effects on bone marrow (BM) and circulating numbers of blood cells. Multiple line-age cytopenias (bi- and pancytopenia) are a frequent sequel of viral infections [1]. Different viruses can directly infect BM stromal cells or haematopoietic stem cells and/or modulate cytokine production by accessory cells. Infections with Gram-negative, LPS-positive bacteria result in an endotoxin-mediated, acute phase, proinflammatory cascade of events that result in granulocyte efflux from the BM, peripheral neutrophilia and BM hyperplasia [2]. In contrast, gram-negative, LPS-negative bacteria, including members of the Anaplasmataceae family, elicit a very different, poorly-characterised, cascade of events. Haematologic alterations in both natural and experimental infection include a marked thrombocytopenia, moderate leukopenia (lymphopenia and neutropenia) and mild to moderate, non-regenerative anaemia [3,4]. Cytopenias vary from profound and irreversible (for example, dogs with chronic E. canis infection) to moderate and transient. Paradoxically, although Anaplasma family organisms display cell tropism for specific haematopoietic cell lineages, infection, regardless of aetiologic agent, host species or cell target, is characterised by bicytopenia or, occasionally, pancytopenia. This suggests the potential for more global haematopoietic disturbances. We hypothesised that the cytopenias may be due in part to bacteria-induced haematopoietic suppression. C57/Bl6 mice were infected intraperitoneally with E. muris and samples were collected on days 0–60 post-infection. C3H/HeN mice were infested with A. phagocytophilum-infected Ixodes scapularis nymphal ticks and sampled on days 0–21 post-infection. Blood samples were collected for a complete blood cell analysis and quantitative PCR for pathogen detection (A. phagocytophilum p44 DNA). BM was collected for haematopoietic colony-forming assays, cytology, histology and quantitative PCR. Cultured BM supernatants from A. phagocytophilum infected mice were collected for cytokine analysis. Thrombocytopenia and anaemia (bicytopenia) developed in both E. muris and A. phagocytophilum-infected mice. Leukopenia was variable. A. phagocytophilum infection induced significant decreases in BM proliferation and differentiation from day 4 to day 21 post-infection, concurrent with the cytopenias (Fig. 1). E. muris infection resulted in a similarly dramatic decrease in BM proliferation and differentiation on days 8 to 14 post-infection. Decreases in BM haematopoietic production included all lineages. Haematopoietic colony forming assays are supplemented with cytokines and assess haematopoietic stem cell/progenitor cell activity in the absence of stromal cells. As such, findings support infection-induced alterations in stem cell differentiation, proliferation or mobilisation. Both thrombocytopenia and myelosuppression were more severe in mice infected with A. phagocytophilum via tick bite compared with mice infected intraperitoneally in previous studies. Thrombocytopenia did not correlate with pathogen burden in blood, as assessed by quantitative PCR, but was strongly correlated with BM colony numbers in A. phagocytophilum-infected mice. This temporal association suggests that rapid changes in BM may be contributing to alterations in circulating platelet numbers. Cytologic evaluation of BM revealed infection-induced lymphocyte depletion and a reciprocal granulocytic hyperplasia in both infection models, similar to kinetics noted in other models of inflammation [5]. In addition, A. phagocytophilum infection resulted in BM production of myelosuppressive chemokines, including the murine interleukin-8 homologues, macrophage inflammatory protein-2 and KC. Findings are compatible with previous work that demonstrated a predominance of myelosuppressive chemokines in human BM after A. phagocytophilum infection [6]. Anaplasma family pathogens appear to perturb normal haematopoiesis, resulting in shifts in BM cell populations and transient failure of haematopoietic colony formation, which may contribute to peripheral blood cytopenias. Fig. 1 Bone marrow was collected from Anaplasma phagocytophilum-infected and uninfected mice on days 0–21 after tick-borne infection. An aliquot of BM was plated in duplicate and cultured in complete MethoCult media for 7–10 days (StemCell Technologies) ...

An injured juvenile red-tailed hawk (Buteo jamaicensis) was evaluated at the Veterinary Medical Teaching Hospital at the University of California, Davis. The hawk was quiet, alert, and emaciated, and had a closed comminuted, mid-diaphyseal ulnar fracture. CBC results included heterophilia with a left shift, monocytosis, and increased plasma fibrinogen concentration. The blood smear included rare heterophils containing small, dark blue inclusions approximately 1-2 mum in diameter that ranged from round to coccobacillary in shape and formed variably shaped aggregates; the morphology of the inclusions was suspicious for Chlamydophila or Ehrlichia spp. pathogens. The hawk died, and histopathologic examination of tissues obtained at necropsy found severe multifocal histiocytic and heterophilic splenitis in addition to chronic hepatitis, myocarditis and epicarditis, meningoencephalitis, and airsacculitis. Using immunohistochemistry the presence of Chlamydia/Chlamydophila spp. antigen within multiple tissues was confirmed. Chlamydophila psittaci DNA was demonstrated in whole blood and fresh splenic tissue via real-time PCR. Direct fluorescent antibody staining of air-dried blood smears was positive in rare leukocytes for Chlamydia/Chlamydophila spp. antigen, and immunocytochemical staining of blood smears for Chlamydia/Chlamydophila spp. antigen was focally positive in rare heterophils. These findings may represent the first reported diagnosis of natural avian C. psittaci infection by visualization of organisms in peripheral blood heterophils. Immunocytochemical evaluation of blood smears was valuable in confirming the diagnosis and may be a useful antemortem test to discriminate between bacteria and other inclusions within heterophils.

Objective— To determine the effect of intrathecal amikacin administration and repeated tenovaginocentesis on the total nucleated cell count (TNCC), total protein (TP) concentration and cytologic characteristics of synovial fluid of the equine digital flexor tendon sheath (DFTS). Study Design— Randomized, cross-over experimental design. Animals— Adult horses (n=8). Methods— Synovial fluid was aseptically collected from the DFTS and either 1 mL amikacin sulfate (250 mg/mL) or lactated Ringer's solution (LRS) was injected into the DFTS. Serial synovial fluid samples were obtained at 0, 12, 24, 48, and 72 hours. The opposite treatment was administered to the contralateral DFTS after a washout period of 2 weeks. Results— Treatment increased TP concentration, TNCC, percentage of neutrophils, and neutrophil counts from baseline levels. There was no difference between treatment of the DFTS with amikacin or LRS. Values peaked at 12–24 hours after the initial centesis and then declined toward baseline levels. Conclusions— Injection and repeat centesis of the normal DFTS with 250 mg amikacin or an equivalent volume of LRS resulted in mild increases in synovial fluid analytes from baseline. Synovial inflammation in this study was not accompanied by lameness at the walk and measured analytes returned toward baseline levels within 12–24 hours of first injection. Clinical Relevance— The effect of tenovaginocentesis and intrathecal administration of amikacin or LRS on DFTS synovial fluid values are modest in most horses; however, some horses can develop marked increases in synovial fluid values that may be interpreted as sepsis.

A 4-year-old, male Golden Retriever was presented to the Veterinary Medical Teaching Hospital at the University of California-Davis with a history of lethargy, inappetance, and vomiting. The patient had generalized lymphadenomegaly, marked thrombocytopenia, mild anemia, and moderate hypoalbuminemia. Moderate to marked histiocytic inflammation and lymphocytic-plasmacytic reactivity of the mesenteric, left popliteal, and right mandibular lymph nodes were diagnosed cytologically. Many macrophages contained granular to amorphous material of a uniform blue color, occasionally in morula formation, suggestive of rickettsial organisms. Exposure to raw trout was subsequently documented, leading to a presumptive diagnosis of salmon poisoning disease (SPD). The patient responded quickly to doxycycline therapy for the causative agent of SPD (Neorickettsia helminthoeca). SPD should be considered as a differential diagnosis for a canine patient with clinical signs of vomiting, diarrhea, lethargy, and lymphadenomegaly; laboratory findings of thrombocytopenia and hypoalbuminemia; and potential exposure to raw fish from an endemic area. The cytologic finding of rickettsial inclusions within lymph node macrophages is reportedly seen within a majority of SPD cases and can be valuable in supporting a clinical suspicion of SPD, as it was in this case.

Insertion of the reactive center loop in beta-sheet A in serpins has been typically inferred from the increased stability of the cleaved form to thermal- and urea-induced denaturation. We describe a convenient and rapid fluorescence-based method that differentiates the loop-inserted form from the loop-exposed form in ovalbumin, a prototypic noninhibitory serpin. Recombinant wild-type and R345A ovalbumins in the intact form bind ANS with equilibrium dissociation constants of 116 and 125 microM and a maximal fluorescence increase of 200 and 264%, respectively, in pH 6.8 buffer. Cleavage of the two proteins with porcine pancreatic elastase results in a 1.6- and 2.6-fold increase in the ANS-binding affinity. While cleavage of the reactive center loop in rR345A ovalbumin results in a approximately 200% increase in the ANS fluorescence, the rWT protein exhibits a approximately 50% decrease. Similar experiments with alpha(1)-proteinase inhibitor and antithrombin, two inhibitory serpins that exhibit reactive center loop insertion, show a decrease in ANS fluorescence on cleavage with porcine pancreatic elastase and thrombin, respectively. Denaturation studies in guanidinium hydrochloride indicate that the reactive center loop is inserted in the main body of the serpin in the cleaved form of rR345A mutant, while it is exposed in the cleaved form of rWT ovalbumin. These results demonstrate that ANS fluorescence change is an indicator of the loop-inserted or loop-exposed form in these recombinant ovalbumins, and thus could be advantageously used for probing reactive center loop insertion in ovalbumins. The major increase in fluorescence for the rR345A mutant on cleavage primarily arises from a change in ANS binding rather than from the generation of an additional ANS-binding site.