Alisdair Boag

Assessment of the diagnostic accuracy of circulating natriuretic peptide concentrations to distinguish between cats with cardiac and non-cardiac causes of respiratory distress

OBJECTIVES To determine if serum natriuretic peptide (NP) concentrations could distinguish cardiac from non-cardiac causes of respiratory distress (RD) in cats. ANIMALS Seventy-four cats from 1 university hospital were used. METHODS Serum NP concentrations were measured in 41 cats with non-cardiac respiratory distress (RD-NC) and compared to 33 cats with RD due to congestive heart failure (RD+CHF) using sandwich enzyme immunoassays (ELISA). RESULTS RD-NC cats had lower (P=0.0001) median NT-proANP and NT-proBNP concentrations (614 and 45 fmol/mL, respectively) than RD+CHF cats (1690 and 523 fmol/mL, respectively). The area under the curve was 0.88 and 0.96 for the receiver operating curve analysis of the diagnostic accuracy of NT-proANP and NT-proBNP concentrations to discriminate RD+CHF from RD-NC cats (P=0.036). An optimum cut-off concentration of 986 fmol/mL for NT-proANP and 220 fmol/mL for NT-proBNP accurately discriminated RD-NC from RC+CHF cats with a sensitivity of 93.8% and 93.9% and a specificity of 80.3% and 87.8%, respectively. CONCLUSIONS Serum NP concentrations were different in RD+CHF cats compared to RD-NC cats. Evaluation of circulating NP concentrations may be helpful in the initial approach to cats presenting with respiratory distress, particularly if advances in ELISA technology result in a rapid cage-side test.

MHC class II association study in eight breeds of dog with hypoadrenocorticism.

Canine hypoadrenocorticism is an endocrine disorder characterised by inadequate secretion of steroid hormones from the adrenal glands. Pathology results from immune-mediated destruction of the adrenal cortex, which is similar to that seen in the human Addison’s disease. Both the canine and human diseases have similar clinical presentation, with the diagnosis based on performing a dynamic adrenocorticotropic hormone stimulation test. MHC class II has previously been associated with the human and canine diseases. In the current study, we conducted an MHC class II association study in eight breeds of dog with diagnoses of hypoadrenocorticism. We demonstrated significant differences in dog leukocyte antigen (DLA) haplotype frequencies in six of these breeds: Cocker spaniel, Springer spaniel, Labrador, West Highland white terrier (WHWT), Bearded collie, and Standard poodle. In the Springer spaniel, the DLA-DRB1*015:01--DQA1*006:01--DQB1*023:01 haplotype was significantly associated with disease risk (p = 0.014, odds ratio (OR) = 5.14) and showed a similar trend in the Cocker spaniel. This haplotype is related to one associated with hypoadrenocorticism in the Nova Scotia duck tolling retriever. Similar haplotypes shared between breeds were demonstrated, with DLA-DRB1*001:01--DQA1*001:01--DQB1*002:01 more prevalent in both affected Labrador (p = 0.0002, OR = 3.06) and WHWT (p = 0.01, OR = 2.11). Other haplotypes that have not previously been associated with the disease were identified. The inter-breed differences in DLA haplotypes associated with susceptibility to canine hypoadrenocorticism could represent divergent aetiologies. This could have implications for clinical diagnosis and future comparative studies. Alternatively, it may suggest that the gene of interest is closely linked to the MHC.

A candidate gene analysis of canine hypoadrenocorticism in 3 dog breeds

Canine hypoadrenocorticism is believed to be an immune-related condition. It is rare in the overall dog population but shows a breed-related predisposition with Standard poodles and Portuguese water dogs having a greater prevalence of the condition. It shares many similarities with human primary adrenal insufficiency and is believed to be a naturally occurring, spontaneous model for the human condition. Short haplotype blocks and low levels of linkage disequilibrium in the human genome mean that the identification of genetic contributors to the condition requires large sample numbers. Pedigree dogs have high linkage disequilibrium and long haplotypes within a breed, increasing the potential of identifying novel genes that contribute to canine genetic disease. We investigated 222 SNPs from 42 genes that have been associated or may be implicated in human Addisons disease. We conducted case-control analyses in 3 pedigree dog breeds (Labrador retriever: affected n = 30, unaffected = 76; Cocker Spaniel: affected n = 19, unaffected = 53; Springer spaniel: affected n = 26, unaffected = 46) and identified 8 associated alleles in genes COL4A4, OSBPL9, CTLA4, PTPN22, and STXBP5 in 3 pedigree breeds. Association with immune response genes PTPN22 and CTLA4 in certain breeds suggests an underlying immunopathogenesis of the disease. These results suggest that canine hypoadrenocorticism could be a useful model for studying comparative genetics relevant to human Addisons disease.

Putative candidate genes for canine hypoadrenocorticism (Addison's disease) in multiple dog breeds

CANINE hypoadrenocorticism results from structural damage/functional defects of the adrenal cortex. In human beings, 21 hydroxylase autoantibodies are an early marker for a clinically similar condition (Addisons disease) in 80–90 per cent of patients (Betterle and others 1999, Falorni and others 1995, 1997, Laureti and others 1998), but these antibodies have not been identified in dogs. There are currently no genetic or other biomarkers for the condition in dogs, and diagnosis is based on high adrenocorticotropin (ACTH) and low cortisol levels or impaired cortisol secretory response to synthetic ACTH; however, diagnosis is complicated and often delayed because of the multifaceted clinical presentation. With a timely diagnosis, affected animals can be treated and they can live a relatively normal life, but many dogs are treated incorrectly for more common conditions, reaching ‘Addisonian crisis’ before a diagnosis has been made. Understanding the genetic involvement in canine hypoadrenocorticism could facilitate the development of a genetic test for disease risk that could be used to identify dogs at increased risk for hypoadrenocorticism and enrol them into a screening programme. Furthermore, it could be used to inform breeding strategies and reduce the incidence of hypoadrenocorticism in susceptible dog breeds. Currently, there is limited understanding of the genetic aetiology in dogs or human beings, although the human leucocyte antigen DRB1 is widely accepted as a functional susceptibility locus in the latter with further risk given by the major histocompatibility complex (MHC) class l region (Gombos and others 2007, Erichsen and others …

Single nucleotide polymorphisms in major histocompatibility class II haplotypes are associated with potential resistance to inflammatory bowel disease in German shepherd dogs

German shepherd dogs (GSD) in the UK are at increased risk of developing the Inflammatory Bowel Disaese (IBD). IBD is believed to be a multifactorial immune mediated disease affecting genetically predisposed dogs. The aim of the current study was to investigate whether susceptibility to IBD in GSD is associated with the major histocompatibility complex (MHC) class II locus (Dog Leukocyte Antigen, DLA). Sequence-based genotyping of the three polymorphic DLA genes DLA-DRB1, -DQA1 and -DQB1 was performed in 56 GSDs affected by IBD and in 50 breed-matched controls without any history of gastrointestinal signs. The haplotype DLA-DRB1*015:02-DQA1*006:01-DQB1*023:01 was found to be present only in the control population and was associated with a reduced risk of IBD (P<0.001). In contrast, the haplotype DLA-DRB1*015:01-DQA1*006:01-DQB1*003:01 was associated with IBD (Odds ratio [OR]=1.93, confidence interval [CI]=1.02-3.67, P=0.05). This study has identified an association between DLA-type and canine IBD, supporting the immunogenetic aetiology and immunopathogenesis of this disease.

Circulating natriuretic peptides concentrations in cats with respiratory distress

Adiponectin has been investigated widely due to its association with adiposity and the metabolic syndrome in human beings. Adiponectin circulates as low- (LMW) and high-molecular weight (HMW) multimers and the latter are the more bioactive forms. There are no reports of the relative proportion (distribution) of adiponectin multimers in feline plasma. The aim of this study was to assess the association of dietary nutrient composition, body weight gain, meal feeding, and insulin sensitivity with HMW adiponectin concentration and adiponectin multimer distribution in cats.1 EVALUATION OF FOUR DNA EXTRACTION METHODS FOR THE DETECTION OF TRITRICHOMONAS FOETUS IN FELINE STOOL SPECIMENS BY POLYMERASE CHAIN REACTION. SH Stauffer, AJ Birkenheuer, MG Levy, H Marr, JL Gookin. College of Veterinary Medicine, North Carolina State University, Raleigh, NC. Feces are increasingly recognized as practical samples for molecular diagnosis of infectious disease. Extraction of PCR-quality DNA from feces can be challenging due to co-extraction of PCR inhibitors. Accordingly, we examined the effect of four commercially-available DNA extraction methods on sensitivity of PCR for detection of Tritrichomonas foetus (TF) in naturally-infected and TF-spiked feline stool. Kits evaluated included ExtractMaster Fecal DNA Extraction Kit, Epicentre Biotechnologies (Kit A); QIAamp DNA Stool Mini Kit, Qiagen (Kit B); UltraClean Fecal DNA Kit, MoBio (Kit C); and ZR Fecal DNA Kit, Zymo Research (Kit D). In accordance with manufacturer instructions, DNA was extracted from 180mg (A,B), 50mg (C), 100 & 150mg (D) aliquots of feline feces to which was added 20ml volumes containing 0–10,000 cultured feline TF. Each kit was also used to extract DNA from the feces of each of 10 naturally infected and 10 uninfected cats. DNA was eluted in 300ml (A), 200ml (B), 50ml (C), or 100ml (D) of respective elution buffer. Endogenous PCR inhibitors in extracted DNA was examined by PCR amplification of an 876 bp gene fragment of bacterial 16S rRNA. DNA was then tested by single tube nested PCR for amplification of partial ITS1, 5.8S and ITS2 rRNA genes of TF. Kit D provided the most sensitive detection of TF DNA as expressed by both organisms per DNA extraction and organisms per PCR reaction. To account for differences in DNA concentrations between kits (i.e. fecal sample size and elution volumes), the limit of detection for each kit as expressed by the number of TF per PCR reaction was as follows: Kit B 5 250, Kit A 5 167, Kit C 5 100, Kit D (150mg fecal sample) 5 5, and Kit D (100mg fecal sample) 5 0.5. PCR performed on DNA extracted from cultured TF (no feces) or TF-spiked feces (100mg) using Kit D was positive with as few as 10 TF per extraction. Further, DNA extraction using Kit D could be completed in the shortest time of all kits tested. These studies identify the ZR Fecal DNA Kit as superior to the other kits tested for extraction of PCR-qualityDNA from feline feces. ABSTRACT #2 INVESTIGATION OF ENTEROBACTER CLOACAE INFECTIONS AT A SMALL ANIMAL VETERINARY TEACHING HOSPITAL. JS Weese. University of Guelph, Guelph, Ontario.2 INVESTIGATION OF ENTEROBACTER CLOACAE INFECTIONS AT A SMALL ANIMAL VETERINARY TEACHING HOSPITAL. JS Weese. University of Guelph, Guelph, Ontario. A wide range of pathogens can cause hospital-associated (HA) infections in small animal hospitals. Among these is Enterobacter cloacae, which is one of the most clinically relevant Enterobacter spp and a common cause of HA infection in humans. Recently, multidrug resistance has become a concern, particularly with emergence of extended-spectrum beta-lactamase and extended spectrum cephalosporinase producing strains. An infection control investigation was initiated at the Ontario Veterinary College Teaching Hospital (OVCTH) in the fall of 2007 in response to anecdotal concerns about Enterobacter cloacae infections in hospitalized animals. Enterobacter cloacae was isolated from 45/36719 animals from January 1, 2005 to October 31, 2007, for an overall incidence of 1.2/ 1000 admissions. The monthly incidence rate ranged from 0 to 4.3/ 1000 admissions. Twenty-one (47%) cases were classified as community-associated, while 17 (38%) were hospital associated. Seven (15%) were community-onset but hospital associated, with three of these associated with other veterinary hospitals. There was no increase in the incidence of overall or hospital-associated infections during the study period. The urinary tract was the most common site of infection (n511, 24%). Wound infections (excluding surgical site infections) accounted for 8 (18%) of infections, with superficial and deep surgical site infections accounting for 7 (16%) and organ/space surgical site infections accounting for another 2 cases. Urinary tract infections were most common among animals with CA infection, accounting for 8/21 (38%) cases with wound infections accounting for 4 (19%) cases. Of the 24 cases associated with the OVCTH, 17 (71%) had surgery, 15 (63%) were hospitalized in the intensive care unit, 10 (42%) had indwelling urinary catheters placed, and 20 (83%) had received antimicrobials prior to onset of infection. Risk factors for E. cloacae infection could not be determined because a noninfected control group was not evaluated. Surgical site infections accounted for 9 (38%) HA cases. Overall, only 2/11 (18%) urinary tract infections were associated with prior placement of a urinary catheter. Nine (20%) animals died or were euthanized and E. cloacae was implicated as a causative or contributing factor in 5 (56%) of those cases. Two main antimicrobial phenotype patterns were identified. One (n525) was characterized by susceptibility to fluoroquinolones, tetracycline, and trimethoprim with variable susceptibility to cefoxitin while the other (n514) was characterized by resistance to these antimicrobials. Prior administration of antimicrobials was associated with presence of the more resistant phenotype (P50.044) but there was no association between this phenotype and origin of infection (P50.74) and no increase in the prevalence of this phenotype from 2005 to 2007 (P50.97). Infections with this phenotype were not associated with nonsurvival (P50.74). There was no evidence of a, HA outbreak or increase in prevalence, yet identification of multidrug resistant E. cloacae in both CA and HA infections is concerning and requires ongoing surveillance. ABSTRACT #3 STAPHYLOCOCCUS PSEUDINTERMEDIUS: A NEWLY RECOGNIZED PATHOGEN IN DOGS AND CATS. MC Faires, D Slavic, JS Weese. Ontario Veterinary College, Animal Health Laboratory, University of Guelph, Guelph, Ontario.3 STAPHYLOCOCCUS PSEUDINTERMEDIUS: A NEWLY RECOGNIZED PATHOGEN IN DOGS AND CATS. MC Faires, D Slavic, JS Weese. Ontario Veterinary College, Animal Health Laboratory, University of Guelph, Guelph, Ontario. Staphylococcus intermedius has typically been regarded as the predominant pathogenic Staphylococcus spp in dogs and cats, and a leading cause of skin and soft tissue infections. In 2005, a novel Staphylococcus species, Staphylococcus pseudintermedius, was identified. This organism is closely related to, but distinct from, S. intermedius. Gene-sequence based methods are required to differentiate these two species; however, these techniques are rarely performed in clinical laboratories, and as a result the prevalence and characteristics of S. pseudintermedius are poorly understood. Recent evidence suggests that S. pseudintermedius may actually be the predominant Staphylococcus spp in dogs and cats but misidentified as S. intermedius by diagnostic laboratories. The objective of this study was to use sequence based methods to identify putative S. intermedius isolates from dogs and cats and to evaluate antimicrobial resistance and virulence factors among S. pseudintermedius isolates. Isolates from dogs and cats identified as S. intermedius by conventional laboratory methods were obtained from the University of Guelph Animal Health Laboratory. Isolates were collected in a serial manner without selection. DNA was extracted, sequencing of the sodA gene was performed, and isolates were identified via sequence alignment with reference staphylococcal strains through GenBank (www.ncbi.nlm.nih.gov/blast/BLAST.cgi). Antimicrobial susceptibility testing was performed and PCR was used to identify various virulence factors and antimicrobial genes. A total of 25 isolates were obtained from 21 dogs and 2 cats. Medical records were not available for 2 of the isolates. 25/25 (100%) were identified as S. pseudintermedius Severity of infection ranged from superficial dermatitis to rapidly fatal necrotizing fasciitis with the majority of isolates from otitis externa 9/23 (39.1%) and urinary tract infections 6/23 (26.1%). Antimicrobial susceptibility was as follows: amoxicillin/clavulanate 23/23 (100%), ampicillin 7/ 23 (30.4%), cephalothin 23/23 (100%), clindamycin 18/23 (78.3%), gentamicin 23/23 (100%), tetracycline 18/23 (78.3%), and trimethoprim/sulfa 19/23 (82.6%). Not all antimicrobials were tested for all isolates, based on laboratory protocols regarding antimicrobial panel and site of infection. Inducible resistance to clindamycin was detected by D-test in 1 isolate reported as clindamycin-susceptible (5.6%). Detection of virulence factors and antimicrobial resistance genes is ongoing. This study identified S. pseudintermedius as an important pathogen in dogs and cats, and suggests that S. intermedius may not be a major concern in these species. Further studies are required to evaluate clinically relevant virulence factors to assist in understanding the pathogenesis of disease caused by S. pseudintermedius. 70