Stanley L. Marks

Evaluation of serum feline pancreatic lipase immunoreactivity and helical computed tomography versus conventional testing for the diagnosis of feline pancreatitis

Serum feline trypsinogen-like immunoreactivity (fTLI) concentrations and abdominal ultrasound have facilitated the noninvasive diagnosis of pancreatitis in cats, but low sensitivities (33% and 20-35%, respectively) have been reported. A radioimmunoassay has been validated to measure feline pancreatic lipase immunoreactivity (fPLI), but the assays sensitivity and specificity have not been established. In human beings, the sensitivity of computed tomography (CT) is high (75-90%), but in a study of 10 cats, only 2 had CT changes suggestive of pancreatitis. We prospectively evaluated these diagnostic tests in cats with and without pancreatitis. In all cats, serum was obtained for fTLI and fPLI concentrations, and pancreatic ultrasound images and biopsies were acquired. Serum fPLI concentrations (P< .0001) and ultrasound findings (P = .0073) were significantly different between healthy cats and cats with pancreatitis. Serum fTLI concentrations (P = .15) and CT measurements (P = .18) were not significantly different between the groups. The sensitivity of fTLI in cats with moderate to severe pancreatitis was 80%, and the specificity in healthy cats was 75%. Feline PLI concentrations were both sensitive in cats with moderate to severe pancreatitis (100%) and specific in the healthy cats (100%). Abdominal ultrasound was both sensitive in cats with moderate to severe pancreatitis (80%) and specific in healthy cats (88%). The high sensitivities of fPLI and abdominal ultrasound suggest that these tests should play an important role in the noninvasive diagnosis of feline pancreatitis. As suggested by a previous study, pancreatic CT is not a useful diagnostic test for feline pancreatitis.

Enteropathogenic Bacteria in Dogs and Cats: Diagnosis, Epidemiology, Treatment, and Control

This report offers a consensus opinion on the diagnosis, epidemiology, treatment, and control of the primary enteropathogenic bacteria in dogs and cats, with an emphasis on Clostridium difficile, Clostridium perfringens, Campylobacter spp., Salmonella spp., and Escherichia coli associated with granulomatous colitis in Boxers. Veterinarians are challenged when attempting to diagnose animals with suspected bacterial-associated diarrhea because well-scrutinized practice guidelines that provide objective recommendations for implementing fecal testing are lacking. This problem is compounded by similar isolation rates for putative bacterial enteropathogens in animals with and without diarrhea, and by the lack of consensus among veterinary diagnostic laboratories as to which diagnostic assays should be utilized. Most bacterial enteropathogens are associated with self-limiting diarrhea, and injudicious administration of antimicrobials could be more harmful than beneficial. Salmonella and Campylobacter are well-documented zoonoses, but antimicrobial administration is not routinely advocated in uncomplicated cases and supportive therapy is recommended. Basic practices of isolation, use of appropriate protective equipment, and proper cleaning and disinfection are the mainstays of control. Handwashing with soap and water is preferred over use of alcohol-based hand sanitizers because spores of C. difficile and C. perfringens are alcohol-resistant, but susceptible to bleach (1:10 to 1:20 dilution of regular household bleach) and accelerated hydrogen peroxide. The implementation of practice guidelines in combination with the integration of validated molecular-based testing and conventional testing is pivotal if we are to optimize the identification and management of enteropathogenic bacteria in dogs and cats.

Prevalence and Histopathologic Characteristics of Pancreatitis in Cats

Despite the high prevalence of feline pancreatic disease, no detailed description on the histopathologic nature of this disease is currently available in the literature. In this study we characterize the distribution and histopathologic changes commonly found in feline pancreases, correlate the lesions with age and gastrointestinal GI and extra-gastrointestinal disease, and compare the pancreatic lesions in cats with those in humans. The entire pancreas was removed and examined from 115 cats presented for necropsy irrespective of the cause of death. Histologic sections from left limb, right limb, and body were scored for lesions of acute (AP) and chronic pancreatitis (CP) with a scoring system based on similar systems used in human and veterinary literature. The lesions of CP in cats resemble CP in humans, with fibrosis being more prominent than inflammatory changes. Cystic degeneration gradually increased as other lesions of CP were more prominent. A distinct nodular change of zymogen depletion and acinar cell dysplasia not associated with pancreatitis was prominent in 15.6% of the pancreases. Histologically, AP consisted of neutrophilic inflammation associated with interstitial edema and necrosis of mesenteric fat. An overall prevalence of 67%, and 45% in clinically normal animals, was identified. CP was found in 69 (60.0%) pancreases, and 58 (50.4%) had CP only, with a significant correlation between age and occurrence of CP. There was a statistically significant higher prevalence of CP in the left limb in animals with gastrointestinal disease. AP was present in 18 animals (15.7%) of which 7 animals had AP only (6.1%).

A duchenne muscular dystrophy gene hot spot mutation in dystrophin-deficient Cavalier King Charles Spaniels is amenable to exon 51 skipping

Background Duchenne muscular dystrophy (DMD), which afflicts 1 in 3500 boys, is one of the most common genetic disorders of children. This fatal degenerative condition is caused by an absence or deficiency of dystrophin in striated muscle. Most affected patients have inherited or spontaneous deletions in the dystrophin gene that disrupt the reading frame resulting in unstable truncated products. For these patients, restoration of the reading frame via antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach. The major DMD deletion “hot spot” is found between exons 45 and 53, and skipping exon 51 in particular is predicted to ameliorate the dystrophic phenotype in the greatest number of patients. Currently the mdx mouse is the most widely used animal model of DMD, although its mild phenotype limits its suitability in clinical trials. The Golden Retriever muscular dystrophy (GRMD) model has a severe phenotype, but due to its large size, is expensive to use. Both these models have mutations in regions of the dystrophin gene distant from the commonly mutated DMD “hot spot”. Methodology/Principal Findings Here we describe the severe phenotype, histopathological findings, and molecular analysis of Cavalier King Charles Spaniels with dystrophin-deficient muscular dystrophy (CKCS-MD). The dogs harbour a missense mutation in the 5′ donor splice site of exon 50 that results in deletion of exon 50 in mRNA transcripts and a predicted premature truncation of the translated protein. Antisense oligonucleotide-mediated skipping of exon 51 in cultured myoblasts from an affected dog restored the reading frame and protein expression. Conclusions/Significance Given the small size of the breed, the amiable temperament and the nature of the mutation, we propose that CKCS-MD is a valuable new model for clinical trials of antisense oligonucleotide-induced exon skipping and other therapeutic approaches for DMD.

Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs

Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both organisms have been associated with acute and chronic large and small bowel diarrhea, and acute hemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitro antimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concentrations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) and C. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline, tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. All C. difficile isolates tested had an MIC of <or=1 for both metronidazole and vancomycin. Ninety-five percent (124/131) of C. perfringens isolates tested had an MIC for ampicillin of <or=0.125 microg/ml. Two C. perfringens isolates had an MIC of >or=256 microg/ml for both erythromycin and tylosin. A third C. perfringens isolate had an MIC of 32 microg/ml for metronidazole. Based on the results of this study, ampicillin, erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C. perfringens-associated diarrhea, although resistant strains do exist. However, because there is limited information regarding breakpoints for veterinary anaerobes, and because intestinal concentrations are not known, in vitro results should be interpreted with caution.

Ultrasonographic Evaluation of the Muscularis Propria in Cats with Diffuse Small Intestinal Lymphoma or Inflammatory Bowel Disease

BACKGROUND An ultrasonographic pattern of thickened muscularis propria in the small intestine and lymphadenopathy have been associated with gastrointestinal lymphoma and inflammatory bowel disease (IBD) in cats. OBJECTIVES To investigate the association of these imaging biomarkers with IBD and lymphoma in cats. ANIMALS One hundred and forty-two cats with a histologic diagnosis of normal small intestine (SI) (n = 56), lymphoma (n = 62), or IBD (n = 24). METHODS Retrospective case review. Pathology records from 1998-2006 were searched for cats with a diagnosis of normal, IBD, or lymphoma, an ultrasonographic examination < 28 days before surgery, and without ultrasonographic evidence of a mass. Multinomial regression analysis was used to determine the association of imaging biomarkers with disease status. RESULTS Cats with thickening of the muscularis propria detected by ultrasonographic examination were more likely to have lymphoma compared with normal SI cats (odds ratio [OR] = 4.0, 95% confidence interval [95% CI] 1.2-13.1, P = .021) and those with IBD (OR = 18.8, 95% CI 2.2-162.7, P = .008). Histologic samples of cats with muscularis propria thickening were more likely to have disease infiltrates in both the mucosal and submucosal layers (OR = 8.1, 95% CI 1.7-38.4, P = .008) than cats with normal SI. Cats with ultrasonographic evidence of lymphadenopathy were more likely to have a diagnosis of lymphoma (OR = 44.9, 95% CI 5.1-393.0, P = .001) or IBD (OR = 10.8, 95% CI 1.1-106.3, P = .041) than normal SI. Fifty-six of 62 cats had confirmed or presumptive diagnosis of diffuse T-cell lymphoma. CONCLUSIONS AND CLINICAL RELEVANCE Older cats with muscularis layer thickening are more likely to have T-cell lymphoma than IBD. The ultrasonographic pattern is associated with histologic infiltrates in the mucosal and submucosal layers of small intestine. Lymphadenopathy is associated with lymphoma or IBD.

Bacterial-associated diarrhea in the dog: a critical appraisal.

The clinical documentation of enteropathogenic bacteria causing diarrhea in dogs is clouded by the presence of many of these organisms existing as normal constituents of the indigenous intestinal flora. The diagnosis of a putative bacterial enteropathogen(s) in dogs should be made based on a combination of parameters, including signalment and predisposing factors, clinical signs, serologic assays for toxins, fecal culture, and PCR. Relying on results of fecal culture alone is problematic, because C perfringens, C difficile, Campylobacter spp, and pathogenic and non-pathogenic E coli are commonly isolated from apparently healthy dogs [10,13,33]. Nevertheless, culture may be useful in procuring isolates for the application of molecular techniques, such as PCR, for detection of specific toxin genes or molecular typing of isolated strains to establish clonality in suspected outbreaks. The oversimplistic attempt to characterize bacterially associated diarrhea by anatomic localization of clinical signs should be discouraged, because most of the previously mentioned bacteria have been associated with small and large intestinal diarrhea. Accurate diagnosis of infections may require diagnostic laboratories to incorporate PCR-based assays using genus- and species-specific primers to facilitate detection of toxin genes and differentiation of species that appear phenotypically and biochemically similar. There has been tremendous interest in the application of microarray technology for the simultaneous detection of thousands of genes or target DNA sequences on one glass slide. This powerful tool could be used for detection of specific pathogenic bacterial strains in fecal specimens obtained from dogs in the future.

A Multi-Institutional Study Evaluating the Diagnostic Utility of the Spec cPL™ and SNAP® cPL™ in Clinical Acute Pancreatitis in 84 Dogs

BACKGROUND Pancreas-specific lipase is reported to aid in diagnosing acute pancreatitis (AP) in dogs but has not been rigorously evaluated clinically. HYPOTHESIS/OBJECTIVES To describe variability of disease in dogs with suspected clinical AP, and to evaluate accuracy of 2 pancreatic-specific lipase immunoassays, Spec cPL (SPEC) and SNAP cPL (SNAP), in diagnosing clinical AP. We hypothesized that SPEC and SNAP provide better diagnostic accuracy than serum amylase or total lipase. ANIMALS A total of 84 dogs; 27 without AP and 57 with clinical signs associated with AP. METHODS Multicenter study. Dogs were prospectively enrolled based upon initial history and physical examination, then retrospectively classified into groups according to the likelihood of having clinical AP by a consensus of experts blinded to SPEC and SNAP results. Bayesian latent class analyses were used to estimate the diagnostic accuracy of SPEC and SNAP. RESULTS The estimates for test sensitivities and specificities, respectively, ranged between 91.5-94.1% and 71.1-77.5% for SNAP, 86.5-93.6% and 66.3-77.0% for SPEC (cutoff value of 200 μg/L), 71.7-77.8% and 80.5-88.0% for SPEC (cutoff value of 400 μg/L), and were 52.4-56.0% and 76.7-80.6% for amylase, and 43.4-53.6% and 89.3-92.5% for lipase. CONCLUSIONS AND CLINICAL IMPORTANCE SNAP and SPEC have higher sensitivity for diagnosing clinical AP than does measurement of serum amylase or lipase activity. A positive SPEC or SNAP has a good positive predictive value (PPV) in populations likely to have AP and a good negative predictive value (NPV) when there is low prevalence of disease.

Circovirus in Tissues of Dogs with Vasculitis and Hemorrhage

We characterized the complete genome of a novel dog circovirus (DogCV) from the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis. DogCV was detected by PCR in fecal samples from 19/168 (11.3%) dogs with diarrhea and 14/204 (6.9%) healthy dogs and in blood from 19/409 (3.3%) of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. Co-infection with other canine pathogens was detected for 13/19 (68%) DogCV-positive dogs with diarrhea. DogCV capsid proteins from different dogs varied by up to 8%. In situ hybridization and transmission electron microscopy detected DogCV in the lymph nodes and spleens of 4 dogs with vascular compromise and histiocytic inflammation. The detection of a circovirus in tissues of dogs expands the known tropism of these viruses to a second mammalian host. Our results indicate that circovirus, alone or in co-infection with other pathogens, might contribute to illness and death in dogs.

Neurotoxicosis in 4 Cats Receiving Ronidazole

T ritrichomonas foetus is a protozoan that can cause chronic large-bowel diarrhea in cats. Ronidazole, a nitroimidazole, has been shown to effectively treat T foetus in cats at doses of 30 to 50 mg/kg body weight twice daily for 14 days. To our knowledge, adverse effects in cats have not been reported. This case series describes neurologic abnormalities suspected to be secondary to ronidazole administration in 4 cats. Case 1 is a 2.5-year-old male castrated Domestic Shorthair that was presented with a chief complaint of chronic, intermittent, mucohemorrhagic diarrhea of 1.5 years’ duration. The owner was unable to comment about the frequency or the urgency of defecation. The cat, when 6 weeks of age, was adopted by the owner from a rescue organization and had been housed strictly indoors since then. He resided with 5 other cats, all of whom tested negative for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) and had no history of diarrhea. The only medical problems among the cats were occasional upper respiratory tract infections. Serum biochemistry panel, CBC, and T4; a blood smear to scan for Mycoplasma hemofelis; and (ELISA) for FeLV antigen and FIV antibody (FeLV/FIV test), performed by the referring veterinarian (rDVM) were unremarkable. Coronavirus 7B antibody titers were 1 : 80, 1 : 160, and 1 : 80 on 3 sequential serum samples in an 8-week period. Initial physical examination (PE) revealed a bright, alert, responsive, and well-hydrated cat. He weighed 4.7 kg, with a body condition score (BCS) of 7 of 9. Rectal temperature was 102.9uF, and a small amount of frank blood was observed on the thermometer. The patient had a mild serous ocular and nasal discharge, as well as gingivitis, faucitis, and halitosis. The remainder of his PE was unremarkable. Serum chemistry profile, CBC, urinalysis, abdominal ultrasound, and FeLV/FIV test were all unremarkable. Fecal flotation and direct wet preparation were negative for intestinal parasite ova and motile protozoa; Giardia ELISA was negative; stained direct preparations were unremarkable; and fecal cultures for Salmonella, Yersinia, and Campylobacter were negative. A rectal scrape revealed neutrophilic inflammation, with occasional cocci, diplococci, and a few sporulating bacteria consistent with Clostridium sp. A second fecal wet smear was positive for flagellated protozoa. Culture for Tritrichomonas foetus was positive, and the patient was prescribed ronidazole at a dose of 54 mg/kg PO q12h for 14 days. Five days after starting ronidazole, the owner reported that the cat had normal stools, lethargy, a blank stare, and appeared to be in ‘‘slow motion.’’ Neurologic evaluation of the cat was declined, and the ronidazole was continued for an additional 3 days. These clinical signs progressed, and the cat also developed an unsteady gait, pelvic-limb weakness, and trembling. Ronidazole therapy was discontinued, and the cat remained stable over several days, with gradual improvement. The patient was presented 7 days after discontinuation of ronidazole, and physical examination revealed a crouching, wide-based stance and mild ataxia in the pelvic limbs. The rest of the physical examination was unremarkable. The cat had complete resolution of its neurologic signs 1 month after discontinuation of ronidazole, and physical examination was unremarkable. The hematochezia recurred several days after discontinuing the ronidazole. Direct wet preparation of feces for motile protozoa and culture for T foetus were negative; however, the owner elected to re-treat with ronidazole at a lower dose (30 mg/kg PO q12h). The hematochezia resolved quickly, but, 12 days later, the owner reported that the cat had been unsteady, stumbling, and ‘‘spacey’’ for several days and that she had been giving him only 1 dose daily during this period. Neurologic signs resolved over a period of weeks after discontinuation of ronidazole. The patient was returned for reevaluation approximately 6 weeks after discontinuing the second course of ronidazole, and the PE was unremarkable. Culture of fresh feces was negative for T foetus. Case 2 is a 5-year-old female spayed Persian who was examined because of chronic mucohemorrhagic diarrhea since the cat was acquired from a breeder at 4 months of age. The diarrhea episodes initially occurred 6 to 8 times daily, and the cat was fecally incontinent. Routine deworming by the rDVM with pyrantel pamoate and empiric antimicrobial administration with metronidazole had no effect on the diarrhea. Dietary trials with Royal Canin Innovative Veterinary Diet Green Peas and Venison, Hill’s Prescription diet w/d, and Nestle Purina OM also had no effect on the diarrhea. The rDVM obtained multiple full-thickness biopsy specimens from the small and large intestines, which were characterized by mild lymphoplasmacytic inflammation. The cat was From the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL (Rosado, Specht); and Department of Medicine and Epidemiology, University of California at Davis School of Veterinary Medicine, Davis, CA (Marks). Reprint requests: Dr. Andrew Specht, P.O. Box 100126, Gainesville, FL, 32610; e-mail: SpechtA@mail.vetmed.ufl.edu.