Introduction: Highly pathogenic Asian H5-subtype avian influenza viruses have been found in poultry and wild birds worldwide since they were first detected in southern China in 1996. Extensive control efforts have not eradicated them. Vaccination prevents such viruses infecting poultry and reduces the number lost to compulsory slaughter. The study showed the efficacy of inactivated H5 vaccine from the H5N8 virus against highly pathogenic H5N8 and H5N6 avian influenza viruses in chickens. Material and Methods: Reverse genetics constructed an H5 vaccine virus using the HA gene of the 2014 H5N8 avian influenza virus and the rest of the genes from A/PR/8/34 (H1N1). The vaccine viruses were grown in fertilised eggs, partially purified through a sucrose gradient, and inactivated with formalin. Chickens were immunised i.m. with 1 µg of oil-adjuvanted inactivated H5 antigens. Results: Single dose H5 vaccine recipients were completely protected from lethal infections by homologous H5N8 avian influenza virus and shed no virus from the respiratory or intestinal tracts but were not protected from lethal infections by heterologous H5N6. When chickens were immunised with two doses and challenged with homologous H5N8 or heterologous H5N6, all survived and shed no virus. Conclusion: Our results indicate that two-dose immunisations of chickens with H5 antigens with oil adjuvant are needed to provide broad protection against different highly pathogenic H5 avian influenza viruses.

OBJECTIVE To determine changes in dimensions of feline skin samples as a result of histologic processing and to identify factors that contributed to changes in dimensions of skin samples after sample collection. SAMPLE Cadavers of 12 clinically normal cats. PROCEDURES Skin samples were obtained bilaterally from 3 locations (neck, thorax, and tibia) of each cadaver; half of the thoracic samples included underlying muscle. Length, width, and depth were measured at 5 time points (before excision, after excision, after application of ink to mark tissue margins, after fixation in neutral-buffered 10% formalin for 36 hours, and after completion of histologic processing and staining with H&E stain). Measurements obtained after sample collection were compared with measurements obtained before excision. RESULTS At the final time point, tissue samples had decreased in length (mean decrease, 32.40%) and width (mean decrease, 34.21%) and increased in depth (mean increase, 54.95%). Tissue from the tibia had the most shrinkage in length and width and that from the neck had the least shrinkage. Inclusion of underlying muscle on thoracic skin samples did not affect the degree of change in dimensions. CONCLUSIONS AND CLINICAL RELEVANCE In this study, each step during processing from excision to formalin fixation and histologic processing induced changes in tissue dimensions, which were manifested principally as shrinkage in length and width and increase in depth. Most of the changes occured during histologic processing. Inclusion of muscle did not affect thoracic skin shrinkage. Shrinkage should be a consideration when interpreting surgical margins in clinical cases.

The aim of this study was to quantify the longitudinal shrinkage of canine small intestinal specimens after resection and fixation in 10% formalin. Samples were obtained from 12 clinically normal dogs of medium to large breed via ventral midline coeliotomy and enterectomy. The length of each sample was measured before excision, immediately after excision, and after 24 h in 10% formalin. The results were interpreted with the use of single-sample t-tests of the average changes; P-values of less than 0.01 were considered significant. The samples indicated a significant decrease in length after resection and fixation. The mean shrinkage from the pre-excision state was 28.3% immediately after excision (P < 0.0001) and 26.3% after 24 h of fixation (P < 0.0001). There was a small but not significant increase in the length of the specimens between the 2nd and 3rd measurement points. Quantification of the longitudinal shrinkage of resected intestinal specimens may improve interpretation of the distance of surgical margins from abnormal tissue in histopathology reports and allow investigation of the margins required for the clearance of specific tumors.

OBJECTIVE To assess differences in skin shrinkage between grossly visible tumor and grossly normal marginal skin of dogs for cutaneous mast cell tumors (MCTs) excised with curative intent and to determine an equation to estimate postexcisional gross tumor margins from preexcisional measurements and vice versa. SAMPLE 19 cytologically confirmed and surgically excised cutaneous MCTs obtained from dogs. PROCEDURES Tumors were measured in craniocaudal and dorsoventral directions before excision, immediately after excision, and after fixation in formalin. Both grossly visible tumor and surrounding grossly normal skin that comprised the surgical margin were measured at each time point. Percentage of shrinkage was compared among time points and between the tumor and surrounding grossly normal skin. Patient and histopathologic variables were correlated to skin shrinkage. RESULTS Overall shrinkage was 17.70%. The amount of shrinkage within the grossly visible tumor (4.45%) was less than that within the surrounding grossly normal skin (24.42%). Most of the shrinkage occurred immediately after excision. There was no effect of age, sex, completeness of excision, or degree of edema. Accuracy of an equation to estimate postexcisional margins from preexcisional measurements was only 18.4%. CONCLUSIONS AND CLINICAL RELEVANCE Grossly evident MCTs of dogs shrunk less than did the grossly normal surrounding skin. Although an equation to estimate postexcisional margins from preexcisional measurements could be derived, it likely would need to contain additional variables not included in the study reported here. Until such an equation exists, care must be used when extrapolating surgical margins from histologic margins and vice versa.

Septicaemic pasteurellosis is a fatal, sometimes epidemic, bacterial disease of domestic and wild animals including deer, bison, elk, and pronghorn antelope caused by Pasteurella multocida. This is the case report of septicaemic pasteurellosisin a captive sambar deer. The carcass was sent from Royal Endurance Stable, Bachok, Kelantan to the Kota Bharu RegionalVeterinary Laboratory for post-mortem. Gross examination of organs was followed by collection of specimens from lung, kidney,liver, spleen and heart for histopathology and bacterial examination. Pooled organ samples with rumen content were collected and sent to the nearest Chemistry Department for investigation. For histology, the liver, lung, spleen, kidney, and heart specimens were fixed in 10% neutral formalin, and routinely embedded in paraffin. Fivemicrometer sections were stained with H&E. Other tests such as worm and ectoparasiteidentification were conducted to identify the parasites. Post-mortem lesions revealed generalised haemorrhage in the organs.Pasteurella multocida serogroup B and E. coli were isolated from multiple tissues of the animal. Histological examination alsorevealed severe congestion and haemorhage of multiple tissues with infiltration of the inflammatory cells. The most likely mode of transmission of these bacteria is through an infected wound and into the bloodstream, thereby causing severe septicemia and death to the animal.

OBJECTIVE To examine the equine foot for the presence of sensory receptors including Merkel cells and small lamellated Pacinian-like corpuscles (SLPCs). SAMPLE Forefeet obtained from 7 horses following euthanasia for reasons other than foot disease. PROCEDURES Disarticulated feet were cut into either sagittal sections or cross sections and immersed in neutral-buffered 4% formalin. Following fixation, samples were obtained from the midline of the dorsal aspect of the hoof wall and from the frog (cuneus ungulae) between the apex and central sulcus. The formalin-fixed, paraffin-embedded hoof wall and frog sections were routinely processed for peroxidase immunohistochemistry and stained with H&E, Alcian blue, and Masson trichrome stains for histologic evaluation. RESULTS Sensory myelinated nerves and specific receptors were identified within the epidermal and dermal tissues of the equine foot including the hoof wall laminae, coronet, and frog. Merkel cells were identified with specific antisera to villin, cytokeratin 20, and protein gene product 9.5 in coronet epidermis and hoof wall. These cells were interspersed among basilar keratinocytes within the frog, coronary epidermis, and secondary epidermal laminae. The SLPCs were present within the superficial dermis associated with the central ridge of the frog (ie, frog stay). Numerous S100 protein and protein gene product 9.5 immunoreactive sensory nerves in close proximity to these receptors were present throughout the dermal tissues within both the frog and hoof wall. CONCLUSIONS AND CLINICAL RELEVANCE The presence of Merkel cells and SLPCs that are known to detect tactile and vibrational stimuli, respectively, further defined the diverse range of neural elements within the equine foot.

OBJECTIVE To describe histologic changes in the temporomandibular joints (TMJs) of horses of various ages. SAMPLE 22 TMJs from cadavers of 11 horses. PROCEDURES Horses were categorized into 3 age groups (group 1, 2 to 10 years old [n = 3]; group 2, 11 to 20 years old [3]; and group 3, > 20 years old [5]). Each TMJ was sectioned into 5-mm slices, preserved in formalin, decalcified in formic acid, and routinely processed for histologic analysis. Joints were systematically assessed by use of previously described methods. Multilevel mixed-effects models were used to examine the data. RESULTS The number of changes was significantly fewer and degree of changes was significantly less within the TMJs of group 1 horses, compared with those of group 3 horses. Comparison among groups revealed that the combination of temporal and mandibular scores for group 1 was significantly lower than for groups 2 or 3. Disk score did not differ significantly between groups 1 and 2, but disk scores of groups 1 and 2 were significantly lower than the disk score of group 3. CONCLUSIONS AND CLINICAL RELEVANCE The assessed lesions were associated with osteoarthritis, and they accumulated in the TMJs as horses aged. In the absence of signs of pain manifested as changes in mastication, behavior, or performance, it would be difficult to determine the point at which accrued pathological changes represented the onset of clinically important osteoarthritis of the TMJs.

OBJECTIVE To determine brain region affinity for and retention of gadolinium in dogs after administration of gadodiamide and whether formalin fixation affects quantification. ANIMALS 14 healthy dogs. PROCEDURES 13 dogs received gadodiamide (range, 0.006 to 0.1 mmol/kg, IV); 1 control dog received a placebo. Dogs received gadodiamide 3 to 7 days (n = 8) or 9 hours (5) before euthanasia and sample collection. Brain regions were analyzed with inductively coupled mass spectrometry (ICP-MS) and transmission electron microscopy. Associations between dose, time to euthanasia, and gadolinium retention quantities (before and after fixation in 5 dogs) were evaluated. RESULTS Gadolinium retention was seen in all brain regions at all doses, except for the control dog. Exposure 3 to 7 days before euthanasia resulted in 1.7 to 162.5 ng of gadolinium/g of brain tissue (dose-dependent effect), with cerebellum, parietal lobe, and brainstem affinity. Exposure 9 hours before euthanasia resulted in 67.3 to 1,216.4 ng of gadolinium/g of brain tissue without dose dependency. Transmission electron microscopy revealed gadolinium in examined tissues. Fixation did not affect quantification in samples immersed for up to 69 days. CONCLUSIONS AND CLINICAL RELEVANCE Gadodiamide exposure resulted in gadolinium retention in the brain of healthy dogs. Cerebellum, parietal lobe, and brainstem affinity was detected with dose dependency only in dogs exposed 3 to 7 days before euthanasia. Fixation had no effect on quantification when tissues were immersed for up to 69 days. Physiologic mechanisms for gadolinium retention remained unclear. The importance of gadolinium retention requires further investigation.

OBJECTIVE To evaluate effects of anatomic location, histologic processing, and sample size on shrinkage of excised canine skin samples. SAMPLE Skin samples from 15 canine cadavers. PROCEDURES Elliptical samples of the skin, underlying subcutaneous fat, and muscle fascia were collected from the head, hind limb, and lumbar region of each cadaver. Two samples (10 mm and 30 mm) were collected at each anatomic location of each cadaver (one from the left side and the other from the right side). Measurements of length, width, depth, and surface area were collected prior to excision (P1) and after fixation in neutral-buffered 10% formalin for 24 to 48 hours (P2). Length and width were also measured after histologic processing (P3). RESULTS Length and width decreased significantly at all anatomic locations and for both sample sizes at each processing stage. Hind limb samples had the greatest decrease in length, compared with results for samples obtained from other locations, across all processing stages for both sample sizes. The 30-mm samples had a greater percentage change in length and width between P1 and P2 than did the 10-mm samples. Histologic processing (P2 to P3) had a greater effect on the percentage shrinkage of 10-mm samples. For all locations and both sample sizes, percentage change between P1 and P3 ranged from 24.0% to 37.7% for length and 18.0% to 22.8% for width. CONCLUSIONS AND CLINICAL RELEVANCE Histologic processing, anatomic location, and sample size affected the degree of shrinkage of a canine skin sample from excision to histologic assessment.

Objective: To determine whether angiogenesis and microglial activation were related to seizure-induced neuronal death in the cerebral cortex of Shetland Sheepdogs with familial epilepsy. Animals: Cadavers of 10 Shetland Sheepdogs from the same family (6 dogs with seizures and 4 dogs without seizures) and 4 age-matched unrelated Shetland Sheepdogs. Procedures: Samples of brain tissues were collected after euthanasia and then fixed in neutral phosphate–buffered 10% formalin and routinely embedded in paraffin. The fixed samples were sectioned for H&E staining and immunohistochemical analysis. Results: Evidence of seizure-induced neuronal death was detected exclusively in samples of cerebral cortical tissue from the dogs with familial epilepsy in which seizures had been observed. The seizure-induced neuronal death was restricted to tissues from the cingulate cortex and sulci surrounding the cerebral cortex. In almost the same locations as where seizure-induced neuronal death was identified, microvessels appeared longer and more tortuous and the number of microvessels was greater than in the dogs without seizures and control dogs. Occasionally, the microvessels were surrounded by oval to flat cells, which had positive immunohistochemical results for von Willebrand factor. Immunohistochemical results for neurons and glial cells (astrocytes and microglia) were positive for vascular endothelial growth factor, and microglia positive for ionized calcium–binding adapter molecule 1 were activated (ie, had swollen cell bodies and long processes) in almost all the same locations as where seizure-induced neuronal death was detected. Double-label immunofluorescence techniques revealed that the activated microglia had positive results for tumor necrosis factor-α, interleukin-6, and vascular endothelial growth factor receptor 1. These findings were not observed in the cerebrum of dogs without seizures, whether the dogs were from the same family as those with epilepsy or were unrelated to them. Conclusions and Clinical Relevance: Signs of angiogenesis and microglial activation corresponded with seizure-induced neuronal death in the cerebral cortex of Shetland Sheepdogs with familial epilepsy. Microglial activation induced by vascular endothelial growth factor and associated proinflammatory cytokine production may accelerate seizure-induced neuronal death in dogs with epilepsy.