Advocating for Generalizability: Accepting Inherent Variability in Translation of Animal Research Outcomes.

Advancing scientific discovery requires investigators to embrace research practices that increase transparency and disclosure about materials, methods, and outcomes. Several research advocacy and funding organizations have produced guidelines and recommended practices to enhance reproducibility through detailed and rigorous research approaches; however, confusion around vocabulary terms and a lack of adoption of suggested practices have stymied successful implementation. Although reproducibility of research findings cannot be guaranteed due to extensive inherent variables in attempts at experimental repetition, the scientific community can advocate for generalizability in the application of data outcomes to ensure a broad and effective impact on the comparison of animals to translation within human research. This report reviews suggestions, based upon work with National Institutes of Health advisory groups, for improving rigor and transparency in animal research through aspects of experimental design, statistical assessment, and reporting factors to advocate for generalizability in the application of comparative outcomes between animals and humans.

Dilated cardiomyopathy of possible dietary origin in a cat.

An 11-year-old spayed female domestic shorthaired cat was diagnosed with severe dilated cardiomyopathy (DCM) and congestive heart failure. The cat had been eating cat foods that were high in pulses (e.g. peas, lentils, chickpeas). Neither plasma nor whole blood taurine concentrations were deficient. Primary treatment included furosemide, pimobendan, and clopidogrel, and changing to diets that did not contain pulses (a taurine supplements was not administered). The cat's clinical signs improved, high-sensitivity cardiac troponin I concentrations decreased, and echocardiographic measurements stayed relatively stable for over one year after initiating cardiac medications and changing the diet. Ultimately, the cat was euthanized for worsening congestive heart failure 374 days after the diagnosis of DCM. Infectious disease testing during the time of clinical surveillance was negative. Routine histopathology of the heart was unremarkable, but electron microscopy of the left ventricle showed large numbers of mitochondria of variable size and structure. A moderate number of lamellar bodies and autophagic vacuoles also were noted. This case report illustrates an unusual case of a cat with DCM unrelated to taurine deficiency. The relative roles of diet change, cardiac medications, and a dedicated owner are unclear, but this cat's relatively long survival time is similar to that seen after diet change in dogs and cats with DCM eating high-pulse diets.

The Role of the IACUC in the Design and Conduct of Animal Experiments that Contribute to Translational Success.

Institutional Animal Care and Use Committees (IACUCs) have a mandated role under the Animal Welfare Act and under Public Health Service Policy to assure the ethical and humane use of research animals in experiments conducted in the United States. The IACUC by virtue of its mandated functions is well positioned to help nurture an institutional culture of optimized animal use since this Committee is often responsible in large part for the culture of animal use that evolves within an institution. In addition to fostering a culture of humane care for research animals and a culture of working with the concepts of the 3Rs (refinement, reduction, replacement), the IACUC can help foster a culture of optimized animal use that encourages high quality reproducible studies that contribute to translational success. In part this is achieved when the IACUC is successful in encouraging interdisciplinary collaboration early and often within the animal use community it serves. Unfortunately in some instances the institutional research community may envisage the IACUC as a bureaucratic burden, regulatory necessity, and compliance tool more than a group that enhances the methodology and quality of animal experiments. A well-functioning IACUC should strive to nurture an institutional culture that places value in enhancing the scientific quality of research to help assure the reproducibility of animal studies and translational success of animal models. This is integral to both high quality science as well as excellence in the supporting animal care and use.

Physiological, pharmacological and toxicological considerations of drug-induced structural cardiac injury.

The incidence of drug-induced structural cardiotoxicity, which may lead to heart failure, has been recognized in association with the use of anthracycline anti-cancer drugs for many years, but has also been shown to occur following treatment with the new generation of targeted anti-cancer agents that inhibit one or more receptor or non-receptor tyrosine kinases, serine/threonine kinases as well as several classes of non-oncology agents. A workshop organized by the Medical Research Council Centre for Drug Safety Science (University of Liverpool) on 5 September 2013 and attended by industry, academia and regulatory representatives, was designed to gain a better understanding of the gaps in the field of structural cardiotoxicity that can be addressed through collaborative efforts. Specific recommendations from the workshop for future collaborative activities included: greater efforts to identify predictive (i) preclinical; and (ii) clinical biomarkers of early cardiovascular injury; (iii) improved understanding of comparative physiology/pathophysiology and the clinical predictivity of current preclinical in vivo models; (iv) the identification and use of a set of cardiotoxic reference compounds for comparative profiling in improved animal and human cellular models; (v) more sharing of data (through publication/consortia arrangements) on target-related toxicities; (vi) strategies to develop cardio-protective agents; and (vii) closer interactions between preclinical scientists and clinicians to help ensure best translational efforts.

Characterization of the genetic basis for autosomal recessive hereditary nephropathy in the English Springer Spaniel.

BACKGROUND: Autosomal recessive hereditary nephropathy (ARHN) was diagnosed in 2 English Springer Spaniels (ESS), a breed not previously reported to be affected by hereditary nephropathy (HN). OBJECTIVE: To identify and characterize the genetic cause of ARHN in ESS. ANIMALS: Sixty-three ESS (2 with ARHN, 2 obligate carriers, and 59 others), 2 mixed-breed dogs with X-linked HN, and 2 English Cocker Spaniels (ECS) with ARHN were included. METHODS: ARHN was diagnosed based on transmission electron microscopy and immunostaining of kidney. DNA from affected dogs was screened for the mutation known to cause ARHN in ECS. Quantities of COL4A3, COL4A4, and COL4A5 mRNA transcripts in renal cortex were determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for ARHN-affected dogs and 7 other dogs. The coding regions of COL4A3 and COL4A4 were sequenced for the 2 ARHN-affected ESS and an unaffected dog. Exon 30 of COL4A4 was sequenced for all 63 ESS. RESULTS: qRT-PCR indicated a significant reduction in transcript levels of both COL4A3 and COL4A4 mRNA in the kidney of ARHN-affected ESS. Sequencing identified a single nucleotide substitution in COL4A4 at base 2806 resulting in a premature stop codon. Thirteen of 25 related dogs were identified as carriers. CONCLUSIONS AND CLINICAL IMPORTANCE: A mutation highly likely to cause ARHN in ESS has been identified.

Gene expression analysis of the acute phase response using a canine microarray.

The safety of pharmaceuticals is typically assessed in the dog and rat prior to investigation in humans. As a result, a greater understanding of adverse effects in these preclinical testing species would improve safety assessment. Despite this need, there is a lack of tools to examine mechanisms and identify biomarkers in the dog. To address this issue, we developed an Affymetrix-based oligonucleotide microarray capable of monitoring the expression of thousands of canine genes in parallel. The custom canine array contains 22,774 probe sets, consisting of 13,729 canine and 9045 human-derived probe sets. To improve cross-species hybridization with human-derived probes, the detection region was moved from the variable 3' UTR to the more homologous coding region. Testing of this strategy was accomplished by comparing hybridization of naive dog liver RNA to the canine array (coding region design) and human U133A array (standard 3' design). Although raw signal intensity was greater with canine-specific probe sets, human-derived probes detected the expression of additional liver transcripts. To assess the ability of this tool to detect differential gene expression, the acute phase response was examined in beagle dogs given lipopolysaccharide (LPS). Hepatic gene expression 4 and 24 h post-LPS administration was compared to gene expression profiles of vehicle-treated dogs (n=3/group). Array data was consistent with an acute inflammatory response, with transcripts for multiple cytokines and acute phase proteins markedly induced 4 h after LPS challenge. Robust changes in the expression of transcripts involved with glucose homeostasis, biotransformation, and extracellular matrix remodeling were observed 24 h post-dose. In addition, the canine array identified several potential biomarkers of hepatic inflammation. Strong correlations were found between gene expression data and alterations in clinical chemistry parameters such as serum amyloid A (SAA), albumin, and alkaline phosphatase (ALP). In summary, this new genomic tool successfully detected basal canine gene expression and identified novel aspects of the acute phase response in dog that shed new light on mechanisms underlying inflammatory processes.

Collateral circulation in experimental coarctation of the aorta in minipigs: a possible association with hypertrophied vasa vasorum.

Experimental coarctation of the thoracic aorta was produced in 13 adult Yucatan minipigs by placing a gradually expanding "C-shaped" occluder around the aorta. The aortic constriction was standardized by measuring the blood pressure above the coarctation, with a mean arterial pressure of 165-170mmHg as the target. The pigs were humanely killed after 8 weeks of hypertension. No deaths were associated with constriction of the aorta, and neither rear limb weakness nor neurological dysfunction was noted. The lack of clinical signs in these animals suggested the development of an extensive collateral circulation. Collateral vessels, which were verified angiographically, included intercostal arteries, muscular intercostal arteries, and the internal thoracic artery. In two of the animals in which the thoracic aorta was completely occluded, revascularization also occurred via small arterial channels. Histologically, there was a marked increase in the number of vessels as well as significant hyperplasia and hypertrophy of smooth muscle of adventitial vasa vasorum. These findings support the hypothesis that collateralization of occluded thoracic aortas may be aided by hypertrophy of the vasa vasorum. To our knowledge, these vessels have not previously been shown convincingly by histopathological methods to revascularize occluded large arteries such as the aorta.

Streptococcus agalactiae and Streptococcus difficile 16S-23S intergenic rDNA: genetic homogeneity and species-specific PCR.

Streptococcus difficile is a non-hemolytic Gram-positive bacterial coccus that causes septicemia and meningoencephalitis in farmed tilapia (Oreochromis sp.) and rainbow trout (Oncorhynchus mykiss). Recent studies have demonstrated S. difficile to be a group B, type Ib streptococcus with a whole cell protein electrophoretic profile indistinguishable from S. agalactiae and a biochemical profile similar to that observed for other group B, type Ib streptococci isolated from fish and frogs. The aim of this study was to expand on these findings by comparative nucleic acid sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacers of S. difficile and S. agalactiae. The 97.7% sequence homology identified in these studies supports the taxonomic relationship of these two organisms. The sequence data generated were also used to construct a pair of species-specific PCR primers for use in molecular detection and identification schemes.

Computed tomography of necrotizing meningoencephalitis in 3 Yorkshire Terriers.

A necrotizing meningoencephalitis of Yorkshire terriers has recently been reported in 6 dogs in Switzerland, 1 dog in Japan and 1 dog in the United States. The purpose of this report is to describe the computed tomographic (CT) findings in 3 dogs with this disease, and to correlate the CT abnormalities with the clinical and pathologic findings in each case. Three Yorkshire Terriers between 2 and 10 years old were evaluated. Physical and neurologic examinations, complete blood count (CBC), serum biochemistry profile, cerebrospinal fluid analysis, and CT scan were performed on all 3 dogs. Brainstem auditory evoked responses (BAER) were evaluated for 2 dogs. Two dogs were euthanized at the owners' request and necropsies were performed. Neurologic examination findings were consistent with a multifocal/diffuse encephalitis involving the cerebrum and brainstem in all 3 dogs. Complete blood count and biochemistry profiles were normal. Elevated protein concentration and a mononuclear pleocytosis were demonstrated in 2 of 3 dogs on cerebrospinal fluid evaluation. Multifocal, extensive areas of decreased opacity throughout the cerebral hemispheres, asymmetric ventriculomegaly, and lack of contrast enhancement were appreciated on CT images of all three dogs. No mass effect was seen. These findings correlated well with pathologic findings at necropsy, which included multiple malacic cavitations within the brain, representing areas of locally extensive necrosis. CT abnormalities in combination with signalment, clinical findings and cerebrospinal fluid analysis should facilitate a presumptive diagnosis of Yorkshire Terrier necrotizing meningoencephalitis.

The efficacy of mechanical abrasion and talc slurry as methods of pleurodesis in normal dogs.

OBJECTIVE: To determine the efficacy of mechanical abrasion and talc slurry as methods for pleurodesis in normal dogs. STUDY DESIGN: Experimental study. ANIMALS OR SAMPLE POPULATION: Ten normal beagle dogs. METHODS: Group I dogs had mechanical abrasion (MA) of the pulmonary and costal pleurae performed in one hemithorax with a dry gauze sponge with a median sternotomy approach. Group II dogs had 100 mL of a 1 g talc slurry (TS) administered into one hemithorax through a tube thoracostomy. Administration of the TS was visualized by using video thoracoscopy. All dogs were evaluated at 2, 10, 20, and 30 days postoperatively by means of thoracic radiography and ultrasonographic thoracic wall measurement. The dogs were euthanatized 30 days postoperatively and a gross necropsy was performed. Hemithoraces were assigned a pleurodesis score (0-4) and an obliteration grade (0-6). Tissues were collected for histopathologic examination of pulmonary pleura, costal pleura, and pleural adhesions. Pulmonary and costal pleurae were graded for the degree of fibrosis (0-4). RESULTS: Obliteration grade and costal pleural fibrosis score were significantly higher for the treated sides in the MA dogs compared with the TS dogs. MA Dogs: Mechanical abrasion dogs had pleurodesis, obliteration, and pleural fibrosis scores that were greater on the treated side than the untreated side, however, the differences were not statistically significant. Only two MA dogs had firm adhesion of the pulmonary pleura to the costal pleura in portions of the cranial and middle lung lobes in the treated hemithorax. Thoracic wall surface area covered with adhesions was 15% and 21% in each of these two dogs. The median pulmonary pleural fibrosis score of all MA dogs for the treated hemithorax was 3 compared to 0 on the untreated side. TS Dogs: There was no statistical difference for pleurodesis scores and obliteration grades between the treated and untreated sides. No dogs showed evidence of pulmonary to costal pleural adhesions. Histopathology showed talc crossover into the untreated side in all five dogs. Median pulmonary fibrosis score of the treated hemithorax was 1 compared with 0 on the untreated side. CONCLUSIONS: Neither method of pleurodesis produced sufficient pleural adhesions to obliterate the pleural space. It is possible that the degree of pulmonary pleural fibrosis present in MA dogs may be sufficient to limit air leakage from pulmonary blebs and bullae resulting in successful treatment of spontaneous pneumothorax.

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.

Rectal stricture in an ostrich (Struthio camelus).

Exploratory celiotomy of an 18-mo-old female ostrich (Struthio camelus) with anorexia, lethargy, and constipation of 5 days' duration showed mesenteric volvulus and a focal narrowing of the rectum approximately 28 cm cranial to the cloaca. The prognosis was poor and the animal was euthanized. Necropsy revealed a fibrinonecrotic proctitis and a 3-cm-long circumferential stricture of the rectum. Histologically, the rectal wall at the stricture was deeply effaced by fibrovascular connective tissue with vascular thrombosis and necrotizing vasculitis, and it resembled the lesions in feeder pigs with fibrinonecrotic colitis.

Development of specific nested oligonucleotide PCR primers for the Streptococcus iniae 16S-23S ribosomal DNA intergenic spacer.

Streptococcus iniae is a cause of septicemia, meningoencephalitis, and death in farmed fish and of cellulitis in human beings. A set of nested oligonucleotide PCR primers that specifically amplified a 373-bp subunit from a variety of clinical isolates from farmed fish and human patients were constructed from a 524-bp consensus sequence of the S. iniae 16S-23S ribosomal DNA intergenic spacer.

Evaluation of postoperative peritoneal lavage in standing horses for prevention of experimentally induced abdominal adhesions.

OBJECTIVE: To evaluate the postoperative use of peritoneal lavage for prevention of experimentally induced intraabdominal adhesions in horses. STUDY DESIGN: Areas of serosal abrasion were created on the jejunum of 12 horses. Postoperatively, six horses had peritoneal lavage, and six horses did not (controls). The number of adhesions was determined at necropsy 2 weeks after surgery. ANIMALS OR SAMPLE POPULATION: 12 horses. METHODS: Five sites of jejunal serosal abrasion were created in each horse. A 32 French thoracic catheter was placed into the right ventral aspect of the abdomen before closure of the abdominal incision. Treated horses had abdominal lavage with 10 L of lactated Ringer's solution on four occasions, then catheters were removed from all horses 34 hours after celiotomy. Horses were necropsied at 2 weeks to quantify the number of intraabdominal adhesions. RESULTS: All control horses and one treated horse developed intraabdominal adhesions. The number of adhesions was significantly less (P < .0293) in treated horses. No adverse inflammatory reactions appeared to be associated with repeated peritoneal lavage using lactated Ringer's solution or use of an abdominal drain. CONCLUSIONS: Peritoneal lavage reduced the frequency of intraabdominal adhesions. CLINICAL RELEVANCE: When postoperative adhesions are likely to develop, postoperative peritoneal lavage may decrease the frequency of adhesion formation.

Use of virulent hemorrhagic enteritis virus for the induction of colibacillosis in turkeys.

Three hundred fifty 1-day-old large white turkeys were reared in brooding batteries to 10 days of age, after which they were moved to floor pens on litter. At 7 weeks of age, poults were allotted into four treatment groups as follows: 1) virulent hemorrhagic enteritis virus (HEV) alone (100 turkeys), 2) Escherichia coli alone (100 turkeys), 3) HEV + E. coli (100 turkeys), and 4) negative controls (50 turkeys). HEV was given orally at 7 weeks of age, followed by E. coli challenge in the drinking water 2 days later for 10 consecutive days. All groups were observed daily for mortality, both during and after challenge. Turkeys that died or were moribund were necropsied, and cultures were taken from the liver and bone marrow for bacterial isolation. Total mortality rates were 23% in the HEV + E. coli group, 10% in the HEV-only group, 3% in the E. coli-only group, and 0% in the negative control group. Cumulative mortality values were significantly different from those of the negative controls (P < or = 0.05) for HEV only and the HEV + E. coli group. E. coli was isolated from the liver and bone marrow of almost all turkeys that died.