In Vitro Characteristics of Canine Primary Tracheal Epithelial Cells Maintained at an Air-Liquid Interface Compared to In Vivo Morphology.

Culturing respiratory epithelial cells at an air-liquid interface (ALI) represents an established method for studies on infection or toxicology by the generation of an in vivo-like respiratory tract epithelial cellular layer. Although primary respiratory cells from a variety of animals have been cultured, an in-depth characterization of canine tracheal ALI cultures is lacking despite the fact that canines are a highly relevant animal species susceptible to various respiratory agents, including zoonotic pathogens such as severe acute respiratory coronavirus 2 (SARS-CoV-2). In this study, canine primary tracheal epithelial cells were cultured under ALI conditions for four weeks, and their development was characterized during the entire culture period. Light and electron microscopy were performed to evaluate cell morphology in correlation with the immunohistological expression profile. The formation of tight junctions was confirmed using transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for the junctional protein ZO-1. After 21 days of culture at the ALI, a columnar epithelium containing basal, ciliated and goblet cells was seen, resembling native canine tracheal samples. However, cilia formation, goblet cell distribution and epithelial thickness differed significantly from the native tissue. Despite this limitation, tracheal ALI cultures could be used to investigate the pathomorphological interactions of canine respiratory diseases and zoonotic agents.

A challenging case of lymphoproliferative disease in a cat.

A 7-year-old male castrated Maine Coon cat presented with edema of the right hindlimb and a markedly enlarged right popliteal lymph node. A CBC showed a neutropenia of 1.5 × 103 /µL. Radiographs and ultrasonographic examination were unremarkable. Cytology of the right popliteal lymph node revealed a mixed population of cells, consisting predominantly of medium to large plasmacytoid lymphocytes, low to moderate numbers of well-differentiated plasma cells and low numbers of small lymphocytes. Plasmacytoid lymphocytes had round nuclei with finely stippled chromatin and one prominent round nucleolus. Low numbers of binucleated cells and bizarre mitotic figures, and rare multinucleated cells were observed. Histopathologic examination of the lymph node showed effacement of the normal lymph node architecture by dense sheets of neoplastic cells. Round to polygonal tumor cells of intermediate size had a low to moderate amount of cytoplasm. Round to indented hyperchromatic nuclei were often eccentrically located and contained one distinct nucleolus. Anisocytosis and anisokaryosis were moderate and 21 mitoses/10 high power field (HPF) were present. Congo red staining was negative. High numbers of tumor cells were positive for lambda light chain immunoglobulin; moderate numbers stained positive for MUM-1. A clonal BCR gene rearrangement was detected with an immunoglobulin heavy chain target (IGH), immunoglobulin lambda light chain (IgL), and kappa deleting element (Kde). Differential diagnoses for the lymphoproliferative disease in this cat included lymphoplasmacytic lymphoma and myeloma-related disorder.

Computer-assisted mitotic count using a deep learning-based algorithm improves interobserver reproducibility and accuracy.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intraobserver discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying or classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, computer-assisted review by pathologists may ensure reliability. In the present study, we compared partial (MC-ROI preselection) and full (additional visualization of MF candidates and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole-slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MFs, and improving classification against imposters. The interobserver consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected hotspot MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study demonstrate that computer assistance may lead to more reproducible and accurate MCs in ccMCTs.

Phenotypical peculiarities and species-specific differences of canine and murine satellite glial cells of spinal ganglia.

Satellite glial cells (SGCs) are located in the spinal ganglia (SG) of the peripheral nervous system and tightly envelop each neuron. They preserve tissue homeostasis, protect neurons and react in response to injury. This study comparatively characterizes the phenotype of murine (mSGCs) and canine SGCs (cSGCs). Immunohistochemistry and immunofluorescence as well as 2D and 3D imaging techniques were performed to describe a SGC-specific marker panel, identify potential functional subsets and other phenotypical, species-specific peculiarities. Glutamine synthetase (GS) and the potassium channel Kir 4.1 are SGC-specific markers in murine and canine SG. Furthermore, a subset of mSGCs showed CD45 immunoreactivity and the majority of mSGCs were immunopositive for neural/glial antigen 2 (NG2), indicating an immune and a progenitor cell character. The majority of cSGCs were immunopositive for glial fibrillary acidic protein (GFAP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and Sox2. Therefore, cSGCs resemble central nervous system glial cells and progenitor cells. SGCs lacked expression of macrophage markers CD107b, Iba1 and CD204. Double labelling with GS/Kir 4.1 highlights the unique anatomy of SGC-neuron units and emphasizes the indispensability of further staining and imaging techniques for closer insights into the specific distribution of markers and potential colocalizations.

Discovery of a phylogenetically distinct poxvirus in diseased Crocodilurus amazonicus (family Teiidae).

A novel poxvirus was discovered in Crocodilurus amazonicus (Teiidae) presenting with a debilitating skin disease. The generated first genome sequence of a reptilian poxvirus revealed the closest phylogenetic relationship to avipoxviruses, highlighting potential virus exchanges between avian and reptilian species.

De novo ZIC2 frameshift variant associated with frontonasal dysplasia in a Limousin calf.

BACKGROUND: Bovine frontonasal dysplasias like arhinencephaly, synophthalmia, cyclopia and anophthalmia are sporadic congenital facial malformations. In this study, computed tomography, necropsy, histopathological examinations and whole genome sequencing on an Illumina NextSeq500 were performed to characterize a stillborn Limousin calf with frontonasal dysplasia. In order to identify private genetic and structural variants, we screened whole genome sequencing data of the affected calf and unaffected relatives including parents, a maternal and paternal halfsibling. RESULTS: The stillborn calf exhibited severe craniofacial malformations. Nose and maxilla were absent, mandibles were upwardly curved and a median cleft palate was evident. Eyes, optic nerve and orbital cavities were not developed and the rudimentary orbita showed hypotelorism. A defect centrally in the front skull covered with a membrane extended into the intracranial cavity. Aprosencephaly affected telencephalic and diencephalic structures and cerebellum. In addition, a shortened tail was seen. Filtering whole genome sequencing data revealed a private frameshift variant within the candidate gene ZIC2 in the affected calf. This variant was heterozygous mutant in this case and homozygous wild type in parents, half-siblings and controls. CONCLUSIONS: We found a novel ZIC2 frameshift mutation in an aprosencephalic Limousin calf. The origin of this variant is most likely due to a de novo mutation in the germline of one parent or during very early embryonic development. To the authors' best knowledge, this is the first identified mutation in cattle associated with bovine frontonasal dysplasia.

Amelanotic Malignant Melanoma in a Himalayan Rex Guinea Pig (Cavia porcellus).

A Himalayan Rex guinea pig was presented with a history of nodular, partially ulcerated masses in the subcutis of the left shoulder. Histological examination revealed a garland-like to nodular, infiltrative neoplastic mass of the epidermis and hair follicle epithelium, which obscured the dermoepidermal junction. Neoplastic cells were immunopositive for S100, PNL-2, vimentin and melan-A antigens. No immunolabelling of CD3, CD79, Iba-1 or pancytokeratin was observed. This is the first detailed description of spontaneous amelanotic malignant melanoma in this species.

Emergence and Epidemiology of Bovine Babesiosis Due to Babesia divergens on a Northern German Beef Production Farm.

Babesia divergens, transmitted by the tick Ixodes ricinus, is the most common cause of bovine babesiosis in northern Europe and plays a role as a zoonotic pathogen. However, several studies have indicated a decline of B. divergens prevalence in Europe during the last decades. Here, we investigate the epidemiology of bovine babesiosis on a beef production farm in northern Germany, which had not been affected by babesiosis until an initial outbreak in 2018. In June 2018, 21 adult cattle died, showing classical symptoms of babesiosis. Babesia divergens merozoites were detected in blood smears of clinically affected animals and the species was confirmed by PCR and sequencing of a part of the 18S rRNA gene. In 2018, screening of the farm's entire stock by PCR revealed that Babesia-positive animals were present in only one of five herds grazing on different pastures. In the following year, further babesiosis cases occurred in multiple herds. In March 2020, 95 cattle were tested for anti-B. divergens antibodies and 36 of them (37.89%) had positive titres. To investigate the local Babesia prevalence in ticks, 1,430 questing I. ricinus ticks (555 larvae, 648 nymphs, 227 adults) were collected on the farm's pastures and subjected to PCR for Babesia detection. Babesia divergens DNA could not be detected, but Babesia microti showed an overall prevalence of 0.49% (7/1,430; 0.88% [2/227] of adult ticks, 0.77% [5/648] of nymphs, 0.00% [0/555] of larvae). Babesia venatorum was detected in 0.42% (6/1,430) of ticks (0.44% [1/227] of adult ticks, 0.77% [5/648] of nymphs, 0.00% [0/555] of larvae) and B. capreoli in 0.07% (1/1,430) of ticks (0.00% [0/227] of adult ticks, 0.15% [1/648] of nymphs, 0.00% [0/555] of larvae). Despite the fact that no B. divergens-positive ticks were found, the collected data suggest a geographical spread of the pathogen on the farm. Bovine babesiosis remains a disease of veterinary importance in Europe and may cause considerable economic losses when (re-)emerging in non-endemic areas, especially as awareness for the disease among veterinarians and farmers declines.

Calcinosis in a roe deer fawn (Capreolus capreolus) in northern Germany.

BACKGROUND: Calcinosis has been reported for a broad range of different animals. Causes for calcinosis include metabolic disorders due to kidney failure, intoxication with calcinogenic plants, or iatrogenic overdose of vitamin D. Especially young animals seem to be very susceptible to developing calcinosis. Currently, however, there is a lack of information on calcinosis in wildlife. CASE PRESENTATION: The following case report describes a roe deer fawn admitted to a clinic due to general weakness and myiasis. Plasma levels for creatinine, urea and phosphate were highly elevated, whereas the total calcium level was decreased. Necropsy revealed calcinosis due to calcification in many organs. The reason for calcinosis in this particular case might be kidney failure. Plasma samples from other hunted roe deer fawns also showed high phosphate levels. CONCLUSIONS: Roe deer fawns might be susceptible to calcinosis due to high plasma phosphate, which could be a result of kidney failure or different feed. Further research into calcium and phosphate homeostasis in roe deer is necessary.

Bovine Babesiosis Diagnosed in Formalin-Fixed, Paraffin-Embedded Tissues by Using In Situ Hybridization.

Bovine babesiosis, caused by Babesia divergens, is in general a rare disease in Europe. Nonetheless, local outbreaks can cause severe economic damage, and postmortem identification represents a diagnostic challenge. During a recent outbreak in May 2018 in northern Germany, 21 animals of a herd of 150 cattle died within 40 days having had clinical signs of fever and hemoglobinuria. Gross examination of 4 of the 21 deceased animals revealed a tick infestation, jaundice, and dark brown staining of urine and kidneys. Histologically, there were iron-positive deposits, hyperplasia of the red pulp of the spleen, and centrilobular necrosis of hepatocytes. In several locations, small basophilic granules suggestive of intraerythrocytic parasites were visible in hematoxylin-eosin- and Giemsa-stained sections. Peripheral blood smears from a living cow from the herd and polymerase chain reaction (PCR) of feeding ticks revealed B. divergens infection. In situ hybridization (ISH) was applied on formalin-fixed, paraffin-embedded (FFPE) tissue of the necropsied cattle to confirm babesiosis in these animals postmortem. Digoxigenin-labeled DNA probes were generated based on a specific nucleotide sequence for B. divergens, obtained by PCR and sequencing of DNA isolates from infected Ixodes ricinus ticks from deceased cattle. ISH using these probes allowed postmortem diagnosis of B. divergens infection in routinely fixed FFPE tissues.

Meningioma and associated cerebral infarction in three dogs.

BACKGROUND: In dogs, meningiomas mostly cause chronic progressive clinical signs due to slow tumor growth. CASE PRESENTATION: In contrast, three dogs were presented with the history of chronic generalized tonic-clonic seizures and peracute deterioration with sudden onset of neurological deficits in accordance with an extensive unilateral forebrain lesion. Magnetic resonance imaging examinations of the dogs revealed a well-delineated extraaxial T2W hyperintense mass in the rostral forebrain with homogeneous contrast enhancement. Additionally, an intraaxial, well-demarcated, unilateral lesion was apparent in the parenchyma supplied by the middle cerebral artery. In two cases, necropsy revealed meningothelial meningioma in the rostral fossa and marked eosinophilic neuronal necrosis, a sign of ischemia, focal malacia, edema and gliosis in the temporal lobe and hippocampus because of a focal thrombosis of the middle cerebral artery. In the third case symptomatic treatment resulted in improvement of clinical signs enabling a good quality of life for the patient. CONCLUSIONS: In dogs with structural epilepsy caused by meningioma, acute deterioration of clinical signs can be associated with ischemic infarctions as a potential complication.

Axonopathy and Reduction of Membrane Resistance: Key Features in a New Murine Model of Human GM1-Gangliosidosis.

GM1-gangliosidosis is caused by a reduced activity of ß-galactosidase (Glb1), resulting in intralysosomal accumulations of GM1. The aim of this study was to reveal the pathogenic mechanisms of GM1-gangliosidosis in a new Glb1 knockout mouse model. Glb1-/- mice were analyzed clinically, histologically, immunohistochemically, electrophysiologically and biochemically. Morphological lesions in the central nervous system were already observed in two-month-old mice, whereas functional deficits, including ataxia and tremor, did not start before 3.5-months of age. This was most likely due to a reduced membrane resistance as a compensatory mechanism. Swollen neurons exhibited intralysosomal storage of lipids extending into axons and amyloid precursor protein positive spheroids. Additionally, axons showed a higher kinesin and lower dynein immunoreactivity compared to wildtype controls. Glb1-/- mice also demonstrated loss of phosphorylated neurofilament positive axons and a mild increase in non-phosphorylated neurofilament positive axons. Moreover, marked astrogliosis and microgliosis were found, but no demyelination. In addition to the main storage material GM1, GA1, sphingomyelin, phosphatidylcholine and phosphatidylserine were elevated in the brain. In summary, the current Glb1-/- mice exhibit a so far undescribed axonopathy and a reduced membrane resistance to compensate the functional effects of structural changes. They can be used for detailed examinations of axon-glial interactions and therapy trials of lysosomal storage diseases.

Detection of MERS-CoV antigen on formalin-fixed paraffin-embedded nasal tissue of alpacas by immunohistochemistry using human monoclonal antibodies directed against different epitopes of the spike protein.

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods.

Cryopreservation of Canine Primary Dorsal Root Ganglion Neurons and Its Impact upon Susceptibility to Paramyxovirus Infection.

Canine dorsal root ganglion (DRG) neurons, isolated post mortem from adult dogs, could provide a promising tool to study neuropathogenesis of neurotropic virus infections with a non-rodent host spectrum. However, access to canine DRG is limited due to lack of donor tissue and the cryopreservation of DRG neurons would greatly facilitate experiments. The present study aimed (i) to establish canine DRG neurons as an in vitro model for canine distemper virus (CDV) infection; and (ii) to determine whether DRG neurons are cryopreservable and remain infectable with CDV. Neurons were characterized morphologically and phenotypically by light microscopy, immunofluorescence, and functionally, by studying their neurite outgrowth and infectability with CDV. Cryopreserved canine DRG neurons remained in culture for at least 12 days. Furthermore, both non-cryopreserved and cryopreserved DRG neurons were susceptible to infection with two different strains of CDV, albeit only one of the two strains (CDV R252) provided sufficient absolute numbers of infected neurons. However, cryopreserved DRG neurons showed reduced cell yield, neurite outgrowth, neurite branching, and soma size and reduced susceptibility to CDV infection. In conclusion, canine primary DRG neurons represent a suitable tool for investigations upon the pathogenesis of neuronal CDV infection. Moreover, despite certain limitations, cryopreserved canine DRG neurons generally provide a useful and practicable alternative to address questions regarding virus tropism and neuropathogenesis.

Co-localization of Middle East respiratory syndrome coronavirus (MERS-CoV) and dipeptidyl peptidase-4 in the respiratory tract and lymphoid tissues of pigs and llamas.

This study investigated the co-localization of the Middle East respiratory syndrome coronavirus (MERS-CoV) and its receptor dipeptidyl peptidase-4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS-CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post-inoculation (p.i.), DPP4-MERS-CoV co-localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co-localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. MERS-CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No MERS-CoV was detected on day 24 p.i. in any tissue by IHC. While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of DPP4 for MERS-CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not DPP4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of DPP4 upon infection.

Intervertebral disc herniation in two coatis (Nasua nasua) and postoperative laminectomy membrane formation.

Magnetic resonance imaging revealed spinal cord compression due to intervertebral disc herniation of Hansen type I and II in the thoracolumbar vertebral column in two middle-aged coatis (Nasua nasua) with chronic progressive paraparesis. Surgical treatment included hemilaminectomy and partial corpectomy in one and dorsal laminectomy in the other coati. Both coatis recovered well after surgery. One showed unremarkable gait 6 and 15 months post surgery, while the other one suffered from recurrence of paraparesis leading to euthanasia because of deterioration of neurological signs 20 months after the first surgery. Necropsy revealed formation of a laminectomy membrane compressing the spinal cord. Histopathological signs of spinal cord injury and findings of degenerative processes in the intervertebral disc were comparable to those described in dogs. In conclusion, this case report shows for the first time that surgical intervention seems to be a useful and safe treatment in chronic intervertebral disc herniation in coatis, but relapses are possible.

Association of Batai Virus Infection and Encephalitis in Harbor Seals, Germany, 2016.

We isolated Batai virus from the brain of a euthanized, 26-year-old, captive harbor seal with meningoencephalomyelitis in Germany. We provide evidence that this orthobunyavirus can naturally infect the central nervous system of a mammal. The full-genome sequence showed differences from a previously reported virus isolate from a mosquito in Germany.

Experimental infection of dromedaries with Middle East respiratory syndrome-Coronavirus is accompanied by massive ciliary loss and depletion of the cell surface receptor dipeptidyl peptidase 4.

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one-third of human patients. Recent data indicate that dromedaries represent an important source of infection, although information regarding viral cell tropism and pathogenesis is sparse. In the current study, tissues of eight dromedaries receiving inoculation of MERS-Coronavirus (MERS-CoV) after recombinant Modified-Vaccinia-Virus-Ankara (MVA-S)-vaccination (n = 4), MVA-vaccination (mock vaccination, n = 2) and PBS application (mock vaccination, n = 2), respectively, were investigated. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence, and scanning electron microscopy. MERS-CoV infection in mock-vaccinated dromedaries revealed high numbers of MERS-CoV-nucleocapsid positive cells, T cells, and macrophages within nasal turbinates and trachea at day four post infection. Double immunolabeling demonstrated cytokeratin (CK) 18 expressing epithelial cells to be the prevailing target cell of MERS-CoV, while CK5/6 and CK14 expressing cells did not co-localize with virus. In addition, virus was occasionally detected in macrophages. The acute disease was further accompanied by ciliary loss along with a lack of dipeptidyl peptidase 4 (DPP4), known to mediate virus entry. DPP4 was mainly expressed by human lymphocytes and dromedary monocytes, but overall the expression level was lower in dromedaries. The present study underlines significant species-specific manifestations of MERS and highlights ciliary loss as an important finding in dromedaries. The obtained results promote a better understanding of coronavirus infections, which pose major health challenges.

Generation and characterization of highly purified canine Schwann cells from spinal nerve dorsal roots as potential new candidates for transplantation strategies.

Schwann cells are promising candidates for transplantation strategies in the central nervous system by promoting axonal regeneration. The dog represents a translational model for human spinal cord injury (SCI) for studies with new repair strategies after intervertebral disk herniation (IVDH). To overcome the necessity for an additional surgical procedure, for the first time a protocol for the isolation and purification of canine Schwann cells from spinal nerve biopsies during standard hemilaminectomy in IVDH-affected paraplegic dogs for potential transplantation has been developed. Purity was assessed by flow cytometry. The results were compared with biopsies from dogs without SCI. Within 26 ± 4 days, 90.2 ± 8.8% p75 neurotrophin receptor (p75NTR )-positive cells were achieved in IVDH dogs. The total cell count in acute/subacute and chronic IVDH (acute/subacute: 6.82 ± 6.36 × 106 ; chronic: 2.29 ± 2.00 × 106 ) differed significantly (p = 0.0120) at the potential time point of transplantation. No differences in culture period and purity were detected between dogs with and without IVDH. Despite the small sample size and the altered environment, the isolation of Schwann cells was successful. Negative influences on isolation and purification due to potential pathological changes at the biopsy site of IVDH-diseased dogs were ruled out by comparison of Schwann cell pellets from diseased and control dogs. Finally, the functionality of Schwann cells from dogs with IVDH was outlined in co-culture experiments with canine dorsal root ganglion neurons. In conclusion, nerve root biopsies provide a sufficient number of highly purified and functional Schwann cells within a useful time period for novel therapeutic strategies in dogs with SCI.

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro.

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research.