Equine alveolar macrophages and monocyte-derived macrophages respond differently to an inflammatory stimulus.

Alveolar macrophages (AMs) are the predominant innate immune cell in the distal respiratory tract. During inflammatory responses, AMs may be supplemented by blood monocytes, which differentiate into monocyte-derived macrophages (MDMs). Macrophages play important roles in a variety of common equine lower airway diseases, including severe equine asthma (SEA). In an experimental model, an inhaled mixture of Aspergillus fumigatus spores, lipopolysaccharide, and silica microspheres (FLS), induced SEA exacerbation in susceptible horses. However, whether equine AMs and MDMs have differing immunophenotypes and cytokine responses to FLS stimulation is unknown. To address these questions, alveolar macrophages/monocytes (AMMs) were isolated from bronchoalveolar lavage fluid and MDMs derived from blood of six healthy horses. Separately, AMMs and MDMs were cultured with and without FLS for six hours after which cell surface marker expression and cytokine production were analyzed by flow cytometry and a bead-based multiplex assay, respectively. Results showed that regardless of exposure conditions, AMMs had significantly higher surface expression of CD163 and CD206 than MDMs. Incubation with FLS induced secretion of IL-1ß, IL-8, TNF-α and IFN-γ in AMMs, and IL-8, IL-10 and TNF-α in MDMs. These results suggest that AMMs have a greater proinflammatory response to in vitro FLS stimulation than MDMs, inferring differing roles in equine lung inflammation. Variability in recruitment and function of monocyte-macrophage populations warrant more detailed in vivo investigation in both homeostatic and diseased states.

Neonatal hyperleukocytosis and regenerative anemia in a septic puppy.

This paper reports a case of neonatal hyperleukocytosis in a dog due to a bacterial infection. A 3-week-old, mixed-breed dog was brought to a veterinary college referral center with a history of weight loss despite a good appetite. Clinical and laboratory examinations included: physical examination, complete blood (cell) count (CBC), serum biochemistry profile, abdominal ultrasound examination, and cytology of liver and bone marrow aspirates. The CBC showed hyperleukocytosis of 158.0 × 109/L (RI: 2.1 to 21.2 × 109/L) and hematocrit of 0.19 L/L (RI: 0.21 to 0.34 L/L). The strong leukemoid reaction was comprised of neutrophils, monocytes, and lymphocytes. The dog was diagnosed with Staphylococcus pseudointermedius liver infection based on liver aspirates and culture. Amoxicillin-clavulanic acid was prescribed. A recheck abdominal ultrasound and CBC repeated 4 wk after initial examination were unremarkable. Neonatal hyperleukocytosis is well-described in human medicine but veterinary studies in small animal neonates are scarce. Key clinical message: Hyperleukocytosis in adult dogs may be caused by leukemia or leukemoid reactions. Generalized sepsis is a leading cause of leukemoid reactions in adult dogs and cats. In puppies, neoplasia is less likely, and other causes should be investigated. Similar to human neonates, puppies can mount a strong leukemoid reaction during an infection, even if it is not a generalized septic process.

Hyperleucocytose néonatale et anémie régénérative chez un chiot septique. Cet article rapporte un cas d'hyperleucocytose néonatale chez un chien dû à une infection bactérienne. Un chien de race mixte âgé de 3 semaines a été amené dans un centre de référence d'une école vétérinaire avec des antécédents de perte de poids malgré un bon appétit. Les examens cliniques et de laboratoire comprenaient : examen physique, numération globulaire complète (CBC), profil biochimique sérique, examen échographique abdominal et cytologie des aspirations du foie et de la moelle osseuse.Le CBC montrait une hyperleucocytose de 158,0 × 109/L (RI : 2,1 à 21,2 × 109/L) et un hématocrite de 0,19 L/L (RI : 0,21 à 0,34 L/L). La forte réaction leucémique était composée de neutrophiles, de monocytes et de lymphocytes. Le chien a été diagnostiqué avec une infection hépatique à Staphylococcus pseudointermedius sur la base d'aspirations et de cultures de foie. L'amoxicilline-acide clavulanique a été prescrit. Une échographie abdominale de contrôle et un CBC répété 4 semaines après l'examen initial étaient sans particularité. L'hyperleucocytose néonatale est bien décrite en médecine humaine mais les études vétérinaires chez les nouveau-nés de petits animaux sont rares.Message clinique clé :L'hyperleucocytose chez les chiens adultes peut être causée par une leucémie ou des réactions leucémiques. La septicémie généralisée est l'une des principales causes de réactions leucémiques chez les chiens et les chats adultes. Chez les chiots, la néoplasie est moins probable et d'autres causes doivent être recherchées. Semblables aux nouveaunés humains, les chiots peuvent développer une forte réaction leucémique lors d'une infection, même s'il ne s'agit pas d'un processus septique généralisé.(Traduit par Dr Serge Messier).

Effects of equine SALSA on neutrophil phagocytosis and macrophage cytokine production.

Salivary scavenger and agglutinin (SALSA) is a secreted protein with various immunomodulatory roles. In humans, the protein agglutinates and inactivates microorganisms, and inhibits the release of pro-inflammatory cytokines. Saliva, which is rich in SALSA, accelerates bacterial phagocytosis, but SALSA's contribution is unclear. In horses, the functions of SALSA in inflammation remain undetermined, so they were investigated through phagocytosis and cytokine assays. Equine SALSA was purified from duodenal tissue, which contains abundant SALSA. To assess phagocytosis, fluorescently-labelled bacteria were incubated with 20, 10, 5, or 2.5 µg/mL of SALSA or phosphate buffered saline (PBS), and then incubated at 37°C or on ice with whole blood from seven healthy horses. Fluorescence was measured by gating on neutrophils using a flow cytometer, and compared between groups. To assess effects on cytokine production, alveolar macrophages were isolated from bronchoalveolar lavage fluid of five healthy horses and cultured in serum-free media for 24 hours with different concentrations of SALSA plus 1 µg/mL lipopolysaccharide (LPS), only LPS, or only media. Cytokines were measured in supernatant using an equine-specific multiplex bead immunoassay. There was significantly greater phagocytosis in samples incubated at 37°C compared to incubation on ice. Samples incubated with 20 µg/mL of SALSA at 37°C had less phagocytosis compared to samples with 10 or 2.5 µg/mL SALSA, or PBS. Alveolar macrophages incubated with SALSA plus LPS released significantly less CXC motif chemokine ligand 1, interleukin-8, interleukin-10, and tumor necrosis factor α, and more granulocyte colony stimulating factor (G-CSF), compared to macrophages incubated with LPS alone. These findings indicate anti-inflammatory effects, which may be due to interference with toll-like receptor 4 recognition of LPS or downstream signaling. Increase in G-CSF following incubation with SALSA suggests a novel mechanism for immunoregulation of alveolar macrophages by SALSA, addressing a knowledge gap regarding its functions in horses.

Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma.

Severe equine asthma (SEA) is a common, debilitating lower airway inflammatory disorder of older horses. Alveolar macrophages (AMs) survey inhaled particulates from barn sources causing them to switch from an anti-inflammatory to a proinflammatory phenotype, resulting in neutrophil recruitment to the lung. This proinflammatory switch may contribute to the development and prolongation of SEA. Validated antibodies to identify the cells involved in the pathogenesis of SEA are lacking. In this study, monoclonal antibodies against CD90, CD163, and CD206 were tested for reactivity with equine leukocytes by immunocytochemistry and flow cytometry. A multi-color flow cytometric assay was developed to identify leukocytes in equine bronchoalveolar lavage fluid (BALF). Four control and 4 SEA-susceptible horses had BALF collected before and after a 48-hour moldy hay challenge. Antibodies against CD90 uniquely labeled equine neutrophils, and antibodies against CD163 and CD206 identified equine macrophages. Postchallenge AM surface expression of CD163 increased in both groups of horses, but the increase was statistically significant in only the SEA-susceptible group (P = .02). The surface expression of CD206 on AMs increased significantly in the SEA-susceptible group (P = .03) but was unchanged in the control group (P = .5). Increased expression of CD163 and CD206 during exacerbation of SEA suggested an association between AM phenotype and lung inflammation. However, functions of AMs in the pathogenesis of SEA remain to be elucidated.

Use of immune repertoire sequencing to resolve discordant microscopic and immunochemical findings in a case of T cell-rich large B cell lymphoma in a young dog.

BACKGROUND: Lymphocytic neoplasms with frequent reactive lymphocytes are uncommonly reported in dogs, and can pose a diagnostic challenge. Different diagnostic modalities such as cytology, flow cytometry, histopathology, immunohistochemistry, and clonality testing, are sometimes required for a diagnosis. This report illustrates the value of using a multi-modal diagnostic approach to decipher a complex lymphocytic tumor, and introduces immune repertoire sequencing as a diagnostic adjunct. CASE PRESENTATION: A 10-month-old Great Dane was referred for marked ascites. Cytologic analysis of abdominal fluid and hepatic aspirates revealed a mixed lymphocyte population including numerous large lymphocytes, yielding a diagnosis of lymphoma. Flow cytometrically, abdominal fluid lymphocytes were highly positive for CD4, CD5, CD18, CD45, and MHC II, consistent with T cell lymphoma. Due to a rapidly deteriorating clinical condition, the dog was euthanized. Post mortem histologic evaluation showed effacement of the liver by aggregates of B cells surrounded by T cells, suggestive of hepatic T cell-rich large B cell lymphoma. Immune repertoire sequencing confirmed the presence of clonal B cells in the liver but not the abdominal fluid, whereas reactive T cells with shared, polyclonal immune repertoires were found in both locations. CONCLUSIONS: T cell-rich large B cell lymphoma is a rare neoplasm in dogs that may be challenging to diagnose and classify due to mixed lymphocyte populations. In this case, the results of histopathology, immunohistochemistry and immune repertoire sequencing were most consistent with a hepatic B cell neoplasm and reactive T cells exfoliating into the abdominal fluid. Immune repertoire sequencing was helpful in delineating neoplastic from reactive lymphocytes and characterizing repertoire overlap in both compartments. The potential pitfalls of equating atypical cytomorphology and monotypic marker expression in neoplasia are highlighted.

Salivary Scavenger and Agglutinin (SALSA) Is Expressed in Mucosal Epithelial Cells and Decreased in Bronchial Epithelium of Asthmatic Horses.

The Salivary Scavenger and Agglutinin (SALSA) protein is an innate immune protein with various alleged functions, including the regulation of inflammation and tissue remodeling. Transcriptomic studies of severe equine asthma (SEA) showed downregulation of the gene encoding SALSA in bronchial epithelium of asthmatic compared to non-asthmatic horses. This study aimed to characterize expression of SALSA in equine tissues by immunohistochemistry (IHC), corroborate potential differences in epithelial gene expression between asthmatic and non-asthmatic horses, and assess the structure of equine SALSA. An antibody against SALSA was validated through immunoprecipitation followed by mass spectrometry and Western blotting to recognize the equine protein. This antibody was applied to tissue microarrays (TMAs) containing 22 tissues each from four horses. A quantitative PCR assay was designed to compare gene expression for SALSA between six asthmatic and six non-asthmatic horses, before and after an asthmatic challenge, using cDNA from endoscopic bronchial biopsies as source material. The SALSA gene from bronchial cDNA samples of 10 horses, was amplified and sequenced, and translated to characterize the protein structure. Immunostaining for SALSA was detected in the mucosal surfaces of the trachea, bronchi, bronchioles, stomach, small intestine and bladder, in pancreatic and salivary gland ducts, and in uterine gland epithelium. Staining was strongest in the duodenum, and the intercalated ducts and Demilune cells of the salivary gland. SALSA was concentrated in the apical regions of the epithelial cell cytoplasm, suggestive of a secreted protein. Gene expression was significantly lower (p = 0.031) in asthmatic compared to non-asthmatic horses. Equine SALSA consisted of three to five scavenger receptor cysteine-rich (SRCR) domains, two CUB (C1r/C1s, uegf, bmp-1) domains and one Zona Pellucida domain. These domains mediate the binding of ligands involved in innate immunity. Varying numbers of SRCR domains were identified in different horses, indicating different isoforms. In summary, equine SALSA has a predilection for mucosal sites, has multiple isoforms, and has decreased expression in asthmatic horses, suggesting alterations in innate immunity in equine asthma.

Canine vector-borne infections in Mauritius.

BACKGROUND: Canine vector-borne diseases have a worldwide distribution, but to the best of our knowledge, no research has been carried out to evaluate their presence on the Indian Ocean island of Mauritius. An investigation into canine vector-borne infections was conducted in dogs (n = 78) resident at an animal shelter in Port Louis, Mauritius using a combination of traditional microscopy and serological methods. METHODS: Ticks were manually collected from the stray dog population for identification as well as for quantifying tick burdens. Blood was also collected from each dog via either the jugular vein or the cephalic vein, and was stored in EDTA tubes. The stored blood was then used to measure PCV values, make blood smears for the identification of parasites, and used for serological testing of vector-borne disease. RESULTS: A total of 178 ticks were collected from 52 dogs and identified as Rhipicephalus sanguineus (175/178) or Amblyomma variegatum (3/178). Twenty-six (33%; 95% CI 23, 45) dogs were seropositive for Ehrlichia spp., and 12 (15%; 95% CI 8, 25) for Anaplasma spp., Dirofilaria antigen was detected in 14 (18%; 95% CI 10, 28), and nine (12%; 95% CI 5, 21) dogs had Hepatozoon canis gamonts observed in blood films during microscopic examination. Eleven (14%; 95% CI 7, 24) dogs were co-infected with two pathogens. Borrelia burgdorferi antibodies were not detected in any dogs. CONCLUSIONS: Infection with these pathogens had no significant effect on the packed cell volume (PCV), but high tick burdens were significantly associated with the presence of a tick-borne pathogen. This is the first study of its kind on the dog population in Mauritius and demonstrates the presence of previously undocumented canine vector-borne infections on the island. The relatively high proportion of infected dogs within the study should alert clinicians to the presence of canine vector-borne diseases on the island of Mauritius.