Angle-stable interlocking nailing in a canine critical-sized femoral defect model for bone regeneration studies: In pursuit of the principle of the 3R's.

Introduction: Critical-sized long bone defects represent a major therapeutic challenge and current treatment strategies are not without complication. Tissue engineering holds much promise for these debilitating injuries; however, these strategies often fail to successfully translate from rodent studies to the clinical setting. The dog represents a strong model for translational orthopedic studies, however such studies should be optimized in pursuit of the Principle of the 3R's of animal research (replace, reduce, refine). The objective of this study was to refine a canine critical-sized femoral defect model using an angle-stable interlocking nail (AS-ILN) and reduce total animal numbers by performing imaging, biomechanics, and histology on the same cohort of dogs. Methods: Six skeletally mature hounds underwent a 4 cm mid-diaphyseal femoral ostectomy followed by stabilization with an AS-ILN. Dogs were assigned to autograft (n = 3) or negative control (n = 3) treatment groups. At 6, 12, and 18 weeks, healing was quantified by ordinal radiographic scoring and quantified CT. After euthanasia, femurs from the autograft group were mechanically evaluated using an established torsional loading protocol. Femurs were subsequently assessed histologically. Results: Surgery was performed without complication and the AS-ILN provided appropriate fixation for the duration of the study. Dogs assigned to the autograft group achieved radiographic union by 12 weeks, whereas the negative control group experienced non-union. At 18 weeks, median bone and soft tissue callus volume were 9,001 mm3 (range: 4,939-10,061) for the autograft group and 3,469 mm3 (range: 3,085-3,854) for the negative control group. Median torsional stiffness for the operated, autograft treatment group was 0.19 Nm/° (range: 0.19-1.67) and torque at failure was 12.0 Nm (range: 1.7-14.0). Histologically, callus formation and associated endochondral ossification were identified in the autograft treatment group, whereas fibrovascular tissue occupied the critical-sized defect in negative controls. Conclusion: In a canine critical-sized defect model, the AS-ILN and described outcome measures allowed refinement and reduction consistent with the Principle of the 3R's of ethical animal research. This model is well-suited for future canine translational bone tissue engineering studies.

Granulomatous encephalomyelitis and intestinal ganglionitis in a spectacled Amazon parrot (Amazona albifrons) infected with Mycobacterium genavense.

An approximately 30-year-old male spectacled Amazon parrot (Amazona albifrons) was presented with a 2-week history of ataxia, head shaking, weight loss and seizures. Gross findings on necropsy examination included atrophy of the musculature, ruffled feathers and minimal epicardial and abdominal fat. Microscopically, there were perivascular cuffs of macrophages with fewer lymphocytes in the grey and white matter of the brain and spinal cord. These lesions were accompanied by gliosis and mild vacuolation of the white matter. In the small intestine, up to 70% of the intestinal ganglia were effaced by infiltrates of macrophages and fewer lymphocytes. The intestinal lamina propria contained multiple inflammatory aggregates of a similar nature. Ziehl-Neelsen staining revealed the presence of numerous bacilli within the cytoplasm of macrophages in the central nervous system (CNS) and enteric ganglia. Amplification of the DNAJ gene confirmed a mycobacterial infection and subsequent polymerase chain reaction (PCR) using a species-specific primer confirmed the aetiology as Mycobacterium genavense. Infection of the CNS with Mycobacterium spp. is uncommon and has not been previously reported in a parrot. This case is unusual in that the organism exhibited tropism for neural tissue.

Coxiella burnetii isolates cause genogroup-specific virulence in mouse and guinea pig models of acute Q fever.

Q fever is a zoonotic disease of worldwide significance caused by the obligate intracellular bacterium Coxiella burnetii. Humans with Q fever may experience an acute flu-like illness and pneumonia and/or chronic hepatitis or endocarditis. Various markers demonstrate significant phylogenetic separation between and clustering among isolates from acute and chronic human disease. The clinical and pathological responses to infection with phase I C. burnetii isolates from the following four genomic groups were evaluated in immunocompetent and immunocompromised mice and in guinea pig infection models: group I (Nine Mile, African, and Ohio), group IV (Priscilla and P), group V (G and S), and group VI (Dugway). Isolates from all of the groups produced disease in the SCID mouse model, and genogroup-consistent trends were noted in cytokine production in response to infection in the immunocompetent-mouse model. Guinea pigs developed severe acute disease when aerosol challenged with group I isolates, mild to moderate acute disease in response to group V isolates, and no acute disease when infected with group IV and VI isolates. C. burnetii isolates have a range of disease potentials; isolates within the same genomic group cause similar pathological responses, and there is a clear distinction in strain virulence between these genomic groups.

Quality of tissue specimens obtained endoscopically from the duodenum of dogs and cats.

OBJECTIVE: To evaluate quality of duodenal tissue specimens obtained endoscopically from dogs and cats and submitted to 1 of 2 diagnostic laboratories for evaluation. DESIGN: Case series. SAMPLE POPULATION: Slides from 50 consecutive canine and 50 consecutive feline endoscopically obtained duodenal tissue specimens submitted to laboratory 1 and 49 consecutive canine and 46 consecutive feline specimens submitted to laboratory 2. PROCEDURE: Slides were examined independently by 3 investigators, and each tissue piece on each slide was classified as clearly inadequate, questionable, or clearly adequate on the basis of 4 criteria. An overall score was then assigned to the slide. RESULTS: Slides from laboratory 1 were more likely to be scored as clearly adequate and less likely to be scored as clearly inadequate than slides from laboratory 2. Clearly adequate slides from laboratory 1 had a higher number of clearly adequate pieces of tissue than did clearly adequate slides from laboratory 2. Slides scored as clearly adequate had a higher number of individual tissue pieces than did slides scored as clearly inadequate. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the quality of endoscopically obtained duodenal tissue specimens submitted to laboratories can vary, possibly because of differences in experience of individuals collecting biopsy specimens. Results suggest that at least 8 individual tissue pieces should be submitted when performing endoscopic biopsy of the duodenum in dogs and cats.

Expression of S100a, vimentin, NSE, and melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of canine melanoma.

We evaluated the expression of vimentin, S100a, and Melan A/MART-1 (melanoma antigen recognized by T cells 1) in seven cell lines established independently from dogs with canine melanoma. We also compared routine immunostaining of 29 clinical specimens from melanoma cases using vimentin, S100a, and neuron-specific enolase (NSE) with staining for Melan A/MART-1 as part of a diagnostic panel. All the cell lines were positive for expression of vimentin and S-100a. MelanA/MART-1 expression was seen consistently in only two of the seven cell lines. Staining for Melan A/MART-1 was most intense near areas of heavy melanin pigmentation. All except one of the clinical specimens were positive for vimentin. S 100a was expressed in the majority of both pigmented (15/20, 75%) and amelanotic (8/9, 88.8%) tumors. Seventeen of 29 (58.6%) tumors were positive for NSE. Melan A/MART-1 was expressed in 18/29 (62%) tumors, including 90% of pigmented tumors, but in no amelanotic tumors. Intensity of Melan A/MART-1 staining correlated positively with biologic behavior, with seven malignant tumors showing negative to weak staining and 10 benign tumors showing moderate to strong staining. Three malignant tumors showed moderate to intense staining for Melan A/ MART-1. Our results suggest that expression of Melan A/MART-1 may be unstable in cultured cell lines. Assessment of both S100a and Melan A/MART-1 expression is useful to confirm a diagnosis of canine melanoma, and Melan A/MART-1 may be especially informative regarding the biologic behavior of these tumors.

Bilateral aural hyalohyphomycosis in sable antelope (Hippotragus niger).

Three juvenile sable antelope (Hippotragus niger) were diagnosed with bilateral aural hyalohyphomycosis based on histopathology. All three animals were suspected to be immunodeficient based on low IgG levels determined using the zinc sulfate turbidity test. The serum and hepatic copper levels of one animal were below the bovine reference range. Clinical signs in the three animals included bilateral ventral deviation of the pinnae with multifocal subcutaneous aural tumefaction and poor body condition. Numerous septate, nonpigmented fungal hyphae were found within the auricular cartilage, dermis, and subcutaneous granulomas. No significant fungal agents were isolated by culture, and no signs of systemic fungal dissemination were identified except for a concurrent fungal rhinitis in one animal.

Bacterial cholecystitis with cardiac and pulmonary dissemination in a blue-naped mousebird (Urocolius macrourus).

A blue-naped mousebird (Urocolius macrourus) was diagnosed by gross necropsy and histopathology as having a chronic, fibrosing bacterial cholecystitis. Acute, severe, necrotizing pneumonia and myocarditis also were present with intralesional gram-negative bacteria. The bacteria within the lungs and heart were suspected to have spread from the biliary tract because of the pattern of distribution and similar gram-staining characteristics. Enterobacter sp. and Escherichia coli were cultured from the pulmonary lesions. Cloacal cultures in clinically normal blue-naped mousebirds and speckled mousebirds (Colius striatus) yielded both Enterobacter sp. and E. coli. We hypothesize a pathogenesis in this bird consisting of biliary stasis of unknown etiology and eventual infection of the biliary tract by the normal gram-negative gastrointestinal flora. Death was believed to be a result of cardiac and respiratory dysfunction secondary to the bacterial dissemination from the biliary tree and endotoxemia.

Intestinal crypt lesions associated with protein-losing enteropathy in the dog.

Six dogs were diagnosed with protein losing enteropathy (PLE). There was no evidence of inappropriate inflammatory infiltrates or lymphangiectasia in multiple mucosal biopsies of the small intestine of 4 of the dogs. The 5th and 6th dogs had obvious lymphangiectasia and a moderate infiltrate of inflammatory cells in the intestinal mucosa. All 6 dogs had a large number of dilated intestinal crypts that were filled with mucus, sloughed epithelial cells, and/or inflammatory cells. Whether PLE occurs in these dogs because of protein lost from the dilated crypts into the intestinal lumen or whether the dilated crypts are a mucosal reaction due to another undetermined lesion that is responsible for alimentary tract protein loss is unknown. However, when large numbers of dilated intestinal crypts are present, they appear to be associated with PLE even if there are no other remarkable lesions in the intestinal mucosa.

Partial tracheal obstruction due to chondromas in ball pythons (Python regius).

Over a 9-mo period, three adult ball pythons (Python regius) (one male, two females) were evaluated for severe dyspnea. Partial obstructions of the tracheal lumen were identified radiographically and/or visualized with a 3.0-mm rigid laparoscope inserted into the tracheal lumen in all three snakes. Administration of systemic antibiotics and nebulization resulted in partial improvement of the dyspnea. In two snakes, the tracheal lesions were removed with a rigid laparoscope and a flexible biopsy instrument inserted into the tracheal lumen. The other snake died and was necropsied. Histologically, the lesions from two snakes were determined to be benign chondromas. The chondromas were composed of a variably disorganized chondroid matrix populated by quiescent, normal-appearing chondrocytes within lacunae, although the chondrocytes were increased in density compared with normal hyaline cartilage and contained rare mitotic figures. The tracheal masses in one snake grew by expansion, not invasion, and were focally continuous with a mineralized cartilage tracheal ring, suggesting a benign nature. This is the second report of tracheal chondroma in ball pythons. Tracheal chondromas are exceedingly rare in humans and domesticated animals, suggesting a possible predisposition of ball pythons for this neoplasm.

Fine-needle aspirate cytology suggesting hepatic lipidosis in four cats with infiltrative hepatic disease.

Four cats are reported in which cytology smears obtained by ultrasound-guided fine needle aspiration of the liver were interpreted as indicative of hepatic lipidosis. However, histopathology of hepatic tissue samples obtained with Tru-Cut-like needles or wedge biopsy revealed that the cats had inflammatory or neoplastic hepatic disease causing their clinical signs. Fine needle aspiration and cytology may not detect infiltrative lesions, particularly those that are nodular, multifocal, or localised around the portal regions. Fine needle aspirate cytology is a useful diagnostic procedure with many advantages, but care must be taken to avoid diagnosing hepatic lipidosis as the cause of illness when an infiltrative lesion is responsible.

Effects of lithium carbonate administration to healthy cats.

Lithium carbonate administration to healthy cats was evaluated in 2 controlled studies (a dose-response study and a bone marrow evaluation study) to determine the effectiveness of lithium as a bone marrow stimulant. Lithium carbonate was administrated at dosage ranging from 300 to 1,050 mg/m2 of body surface/d. Complete blood count, serum lithium concentration determination, serum biochemical analysis, urinalysis, and bone marrow aspiration and biopsy were periodically performed. Serum lithium concentration greater than 2 mEq/L was associated with significant decrease in numbers of circulating segmented neutrophils (less than 1,200 cells/microliter; P less than 0.01) and lymphocytes (less than 1,300 cells/microliter; P less than 0.0001), as well as significant (P less than 0.05) decrease in urine specific gravity. Bone marrow evaluation revealed apparent maturation arrest of the neutrophil cell line. Coincident with the changes in laboratory values, the lithium-treated cats became ill. Changes in behavior and vocalization were seen, followed by anorexia, vomiting, and diarrhea. In later stages of intoxication, cats became hyperexcitable and manifested coarse muscular tremors. It was concluded that lithium carbonate does not have potential value as a bone marrow stimulant and is toxic to cats at serum concentration greater than 2 mEq/L.

Effect of dietary iron content on hematologic and other measures of iron adequacy in dogs.

Eighteen 9- to 10-week old Beagles were fed casein-based diets (4,710 kcal of metabolizable energy/kg of body weight) containing either 12, 80, or 160 mg of iron/kg of diet. Growth and feed consumption were monitored throughout the 47-day study. Hematocrit (Hct), hemoglobin (Hb) concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), RBC numbers, erythrocyte protoporphyrin (EP) concentration, serum iron concentration, serum total iron-binding capacity (TIBC), and serum ferritin concentration were determined weekly. Growth rate and feed efficiency were not significantly influenced by dietary iron content. At 14 days, Hb concentration, Hct, MCV, MCH, RBC numbers, and serum iron concentration were significantly (P less than 0.05) lower in dogs fed the 12 mg/kg diet, and remained significantly low for the remainder of the study. Erythrocyte protoporphyrin concentration increased significantly (P less than 0.05) by 14 days in dogs fed the basal diet, and remained significantly high relative to that in dogs of the other dietary groups for the remainder of the study. Serum ferritin concentration decreased in dogs of the group fed the basal diet, with a significant (P less than 0.05) difference beyond day 42. Differences in Hct, MCH, MCV, or hemoglobin, serum iron, serum ferritin, or EP concentration were not found between groups fed 80 and 160 mg of iron/kg of diet. Liver nonheme iron content was significantly (P less than 0.05) affected by dietary iron content.

Relationship of serum ferritin and iron concentrations and serum total iron-binding capacity to nonheme iron stores in dogs.

The relationships of various iron-related analytes were evaluated in 95 dogs. Liver and spleen nonheme iron content was determined coulometrically on acid-digested tissue specimens. Serum iron concentration and total iron-binding capacity also were measured coulometrically, whereas serum ferritin concentration was measured by ELISA. Significant (P less than 0.0002) correlation was found between serum ferritin concentration and nonheme iron stores. Significant correlation was not found between nonheme iron stores and serum iron concentration or total iron-binding capacity. Serum ferritin concentration should provide a convenient and relatively noninvasive means of estimating iron stores in dogs.

Enzyme-linked immunosorbent assay for canine serum ferritin, using monoclonal anti-canine ferritin immunoglobulin G.

Immunoassay of serum ferritin is currently used to evaluate the clinical iron status of human beings, horses, cattle, and swine. Because ferritins are immunologically species specific, a separate assay must be developed for each species. We have developed an ELISA for serum ferritin in dogs, using a monoclonal anti-canine ferritin antibody. Ferritin standards were linear (r = 0.997) from 0 to 80 ng/ml. Recovery of ferritin from canine serum was 94%. Dilutions of pooled canine serum were linear from 0 to 50% (r = 0.994). Within-assay coefficient of variability was 5.5%, whereas assay-to-assay coefficient of variability ranged from 12.5 to 21%. This assay should provide a nonsurgical means of accurately estimating dogs' iron stores.

Chloramphenicol, lincomycin and oxytetracycline disposition in calves with experimental pneumonic pasteurellosis.

The effects of pneumonia on the pharmacokinetics of chloramphenicol, lincomycin, and oxytetracycline were evaluated in two-month-old calves. Pneumonia was induced by injection of Pasteurella haemolytica cultures directly through the thoracic wall into each lung. Six days prior to induction of pneumonia, the antibiotics were administered in a single i.v. dose. The antibiotics were administered again 48 (i.v.), 60 and 72 h (i.m.) following injection of P. haemolytica. The pharmacokinetics of chloramphenicol (25 mg/kg) and lincomycin (10 mg/kg) were not significantly different in calves with pneumonia. The hybrid rate constant beta for oxytetracycline was increased in calves with pneumonia from 0.0034 +/- 0.0003/min to 0.0048 +/- 0.0007/min between 2 h and 8 h. Thus the elimination half-life in serum was shortened from 212.4 +/- 20.3 min to 149.3 +/- 19.5 min. In addition, there was an apparent but not statistically significant decrease in K12 with pneumonia. These findings accentuate the need for observance of 12-h dose intervals with oxytetracycline.