Joann M. Kinyon

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.

Isolation and Characterization of Methicillin-Resistant Staphylococcus aureus from Pork Farms and Visiting Veterinary Students

In the last decade livestock-associated methicillin-resistant S. aureus (LA-MRSA) has become a public health concern in many parts of the world. Sequence type 398 (ST398) has been the most commonly reported type of LA-MRSA. While many studies have focused on long-term exposure experienced by swine workers, this study focuses on short-term exposures experienced by veterinary students conducting diagnostic investigations. The objectives were to assess the rate of MRSA acquisition and longevity of carriage in students exposed to pork farms and characterize the recovered MRSA isolates. Student nasal swabs were collected immediately before and after farm visits. Pig nasal swabs and environmental sponge samples were also collected. MRSA isolates were identified biochemically and molecularly including spa typing and antimicrobial susceptibility testing. Thirty (30) veterinary students were enrolled and 40 pork farms were visited. MRSA was detected in 30% of the pork farms and in 22% of the students following an exposure to a MRSA-positive pork farm. All students found to be MRSA-positive initially following farm visit were negative for MRSA within 24 hours post visit. Most common spa types recovered were t002 (79%), t034 (16%) and t548 (4%). Spa types found in pork farms closely matched those recovered from students with few exceptions. Resistance levels to antimicrobials varied, but resistance was most commonly seen for spectinomycin, tetracyclines and neomycin. Non-ST398 MRSA isolates were more likely to be resistant to florfenicol and neomycin as well as more likely to be multidrug resistant compared to ST398 MRSA isolates. These findings indicate that MRSA can be recovered from persons visiting contaminated farms. However, the duration of carriage was very brief and most likely represents contamination of nasal passages rather than biological colonization. The most common spa types found in this study were associated with ST5 and expands the range of livestock-associated MRSA types.

In vitro antimicrobial inhibition profiles of Mycoplasma bovis isolates recovered from various regions of the United States from 2002 to 2003

Antimicrobial therapy continues to be important in reducing losses due to pneumonic forms of Mycoplasma bovis disease in beef and dairy calves. Although M. bovis diseases have been documented as frequent and economically important in the United States, there are no published reports on the antimicrobial activity of approved compounds against US strains. In this study, the authors report on the activity of 9 different antimicrobials against 223 recently recovered isolates of M. bovis. These isolates represent accessions from 5 geographic regions of the United States and were grouped by 4 tissues of origin (milk, respiratory, joint, or ear and eye). A broth microdilution test was used to determine minimum inhibitory concentration (MIC) values by reading redox changes detected in broth with alamarBlue (resazurin) indicator. For each antimicrobial, the median, MIC50, MIC90, mode, and range were calculated, and the values used for comparisons. In the absence of accepted breakpoint values, published MIC cutoff values for animal mycoplasmas as well as Clinical Laboratory Standards Institute interpretive criteria were used as a reference to define in vitro activity. The MIC values from active antimicrobials were found to distribute independently of region of origin of the isolates or of tissue of origin. Enrofloxacin, florfenicol, and spectinomycin were found to be active compounds in vitro. Oxytetracycline and chlortetracycline were active against more than half of the isolates. Very few isolates were inhibited by tilmicosin and none by erythromycin, ampicillin, or ceftiofur. The antimicrobial profiles determined for these US strains were remarkably similar to those reported for European isolates. However, unlike in Europe, there appears to be no diversity of profiles when US isolates are grouped by region or tissue of origin.

A Prospective, Case Control Study Evaluating the Association between Clostridium Difficile Toxins in the Colon of Neonatal Swine and Gross and Microscopic Lesions

Clostridium difficile infection in swine has most often been described in suckling pigs, where it has been associated with mesocolonic edema and typhlocolitis. This prospective study was designed to assess the correlation between the presence of C. difficile toxins (TCd) in the colon contents of neonatal pigs and a number of parameters, including gross evidence of diarrhea, mesocoloninc edema, typhlitis, and colitis. C. difficile was isolated from 51% (66/129) of large intestines and TCd was detected in the colon contents of 50% (65/129) of the piglets. Fifty-eight percent (38/65) of TCd-positive piglets had normal to pelleted colon and rectal contents, whereas 75% (48/64) of TCd-negative pigs had gross evidence of diarrhea. Clostridium difficile toxin-positive animals were significantly more likely to have normal to pelleted feces. Edema of the mesocolon was observed in 38/65 (59%) of TCd-positive piglets. Because a high number of TCd-positive piglets (41%) lacked edema of the mesocolon and a high number of TCd-negative pigs had mesocolonic edema (51%), a statistically significant association between TCd and mesocolonic edema was not identified. Seventy-five percent (49/65) of TCd-positive piglets had colitis and 47/65 (72%) had typhlitis. The association between TCd and both colitis and typhlitis was statistically significant. Apparently healthy piglets were obtained from 5 separate sites. Because TCd was detected in the colon contents of 23/29 (79%) apparently healthy piglets obtained from 5 separate sites, and 70% of TCd-positive control pigs had colitis, C. difficile may represent an important subclinical issue in neonatal swine.

Surveillance of Staphylococcus aureus in Veterinary Teaching Hospitals

ABSTRACT Staphylococcus aureus isolates (n = 70) from 65 patients (36 canine, 18 equine, 7 bovine, 2 avian, and 2 feline) at seven veterinary teaching hospitals in the United States were studied. The majority of patients (83%) with an S. aureus infection were canine and equine, but this may have reflected a sample bias based on clinic case loads and diagnostic lab submissions at the participating institutions. Fourteen percent of patients with an S. aureus infection were infected with a methicillin-resistant S. aureus (MRSA) isolate. Six of seven institutions had at least one MRSA infection during the study. Pulsed-field gel electrophoresis on 63 of the 70 isolates yielded 58 unique strains of S. aureus. None of the strain types of the MRSA isolates matched each other or the type of any other S. aureus isolate. The proportions of patients infected with an MRSA isolate were not significantly different between institutions or animal species (P ≥ 0.222). Methicillin-resistant S. aureus isolates in this study seemed to be community acquired rather than hospital acquired.

Development of a Panel of Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of Major Agents Causing Calf Diarrhea in Feces

Calf diarrhea is a major economic burden to the bovine industry. Since multiple infectious agents can be involved in calf diarrhea, and the detection of each of the causative agents by traditional methods is laborious and expensive, a panel of 2 multiplex real-time polymerase chain reaction (PCR) assays was developed for rapid and simultaneous detection of the 5 major bovine enteric pathogens (i.e., Bovine coronavirus [BCoV; formally known as Betacoronavirus 1], group A Bovine rotavirus [BRV], Salmonella spp., Escherichia coli K99+, and Cryptosporidium parvum). The estimated detection limit (i.e., analytic sensitivity) of the panel was 0.1 TCID50 (50% tissue culture infective dose) for BCoV and group A BRV; 5 and 0.5 colony-forming units for E. coli K99+ and Salmonella, respectively; and 50 oocysts for Cryptosporidium per reaction. In testing 243 fecal samples obtained from submissions to the Iowa State University Veterinary Diagnostic Laboratory or from experimental animals with known infection status, the newly developed multiplex realtime PCR panel simultaneously detected all 5 pathogens directly from fecal samples and was more rapid and sensitive than the traditional diagnostic tests. The PCR panel showed 89%–97% agreement with those conventional diagnostic tests, demonstrating diagnostic sensitivity equal to or better than that of the conventional tests. In conclusion, the multiplex real-time PCR panel can be a tool for a timely and accurate diagnosis of calf diarrhea associated with BCoV, group A BRV, E. coli K99+, Salmonella, and/or Cryptosporidium.

Lung Phenotype of Juvenile and Adult Cystic Fibrosis Transmembrane Conductance Regulator–Knockout Ferrets

Chronic bacterial lung infections in cystic fibrosis (CF) are caused by defects in the CF transmembrane conductance regulator chloride channel. Previously, we described that newborn CF transmembrane conductance regulator-knockout ferrets rapidly develop lung infections within the first week of life. Here, we report a more slowly progressing lung bacterial colonization phenotype observed in juvenile to adult CF ferrets reared on a layered antibiotic regimen. Even on antibiotics, CF ferrets were still very susceptible to bacterial lung infection. The severity of lung histopathology ranged from mild to severe, and variably included mucus obstruction of the airways and submucosal glands, air trapping, atelectasis, bronchopneumonia, and interstitial pneumonia. In all CF lungs, significant numbers of bacteria were detected and impaired tracheal mucociliary clearance was observed. Although Streptococcus, Staphylococcus, and Enterococcus were observed most frequently in the lungs of CF animals, each animal displayed a predominant bacterial species that accounted for over 50% of the culturable bacteria, with no one bacterial taxon predominating in all animals. Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry fingerprinting was used to quantify lung bacteria in 10 CF animals and demonstrated Streptococcus, Staphylococcus, Enterococcus, or Escherichia as the most abundant genera. Interestingly, there was significant overlap in the types of bacteria observed in the lung and intestine of a given CF animal, including bacterial taxa unique to the lung and gut of each CF animal analyzed. These findings demonstrate that CF ferrets develop lung disease during the juvenile and adult stages that is similar to patients with CF, and suggest that enteric bacterial flora may seed the lung of CF ferrets.

Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs

Classical swine dysentery is associated with the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae. However, multiple Brachyspira spp. can colonize the porcine colon. Since 2008, several Brachyspira spp. not identified as B. hyodysenteriae by genotypic and/or phenotypic methods have been isolated from the feces of pigs with clinical disease typical of swine dysentery. In the current study, 8 clinical isolates, including 5 strongly beta-hemolytic and 3 weakly beta-hemolytic Brachyspira strains, and a reference strain of B. hyodysenteriae (B204) were inoculated into pigs (n = 6 per isolate) to compare pathogenic potential following oral inoculation. Results revealed that strongly beta-hemolytic isolates induced significantly greater typhlocolitis than those that are weakly beta-hemolytic, regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as “Brachyspira sp. SASK30446” and Brachyspira intermedia by polymerase chain reaction (PCR) produced lesions similar to those caused by B. hyodysenteriae. The results suggest that phenotypic culture characteristics of Brachyspira spp. may be a more sensitive indicator of potential to induce dysentery-like disease in pigs than molecular identification alone based on currently available PCR assays. Additionally, culture of mucosal scrapings obtained at necropsy was more sensitive than direct PCR on the same samples for detection of Brachyspira spp.

Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets.

Cystic fibrosis (CF) is a multiorgan disease caused by loss of a functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in many epithelia of the body. Here we report the pathology observed in the gastrointestinal organs of juvenile to adult CFTR-knockout ferrets. CF gastrointestinal manifestations included gastric ulceration, intestinal bacterial overgrowth with villous atrophy, and rectal prolapse. Metagenomic phylogenetic analysis of fecal microbiota by deep sequencing revealed considerable genotype-independent microbial diversity between animals, with the majority of taxa overlapping between CF and non-CF pairs. CF hepatic manifestations were variable, but included steatosis, necrosis, biliary hyperplasia, and biliary fibrosis. Gallbladder cystic mucosal hyperplasia was commonly found in 67% of CF animals. The majority of CF animals (85%) had pancreatic abnormalities, including extensive fibrosis, loss of exocrine pancreas, and islet disorganization. Interestingly, 2 of 13 CF animals retained predominantly normal pancreatic histology (84% to 94%) at time of death. Fecal elastase-1 levels from these CF animals were similar to non-CF controls, whereas all other CF animals evaluated were pancreatic insufficient (<2 μg elastase-1 per gram of feces). These findings suggest that genetic factors likely influence the extent of exocrine pancreas disease in CF ferrets and have implications for the etiology of pancreatic sufficiency in CF patients. In summary, these studies demonstrate that the CF ferret model develops gastrointestinal pathology similar to CF patients.

In vitro activity of four antimicrobial agents against North American isolates of porcine Serpulina pilosicoli

Porcine colonic spirochetosis is a nonfatal diarrheal disease that affects pigs during the growing and finishing stages of production. The disease is caused by Serpulina pilosicoli, a newly recognized species of pathogenic intestinal spirochete. Antimicrobial therapy aimed at reducing the infection may be helpful in controlling spirochetal diarrhea. In this study, the in vitro antimicrobial susceptibilities of the reference isolate S. pilosicoli P43/6/78 from the United Kingdom and 19 field isolates obtained from pigs in Canada (n = 5) and the United States (n = 14) were determined against the antimicrobial agents carbadox, gentamicin, lincomycin, and tiamulin, all of which are commonly used for control of the related pathogenic intestinal spirochete S. hyodysenteriae. Additionally, the susceptibility or resistance of each isolate against each antimicrobial agent was estimated on the basis of available data on the in vitro antimicrobial susceptibility breakpoints of S. hyodysenteriae. Each isolate was identified on the basis of phenotypic and genotypic markers, and the minimum inhibitory concentration of each antimicrobial agent was determined by the agar-dilution method. All the isolates were susceptible to carbadox and tiamulin. The percentages of isolates susceptible, intermediate, and resistant to lincomycin were 42.1%, 42.1%, and 15.8%, respectively. Slightly less than half of the isolates (47.4%) were susceptible to gentamicin, and the remainder (52.6%) were resistant. Implementation of rational control measures to reduce infection by S. pilosicoli should improve overall health and productivity in swine herds.