Terri Hathcock

Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow

OBJECTIVE Although several types of stem cells have been isolated from rodent and human tissues, very few data exist on stem cell isolation from nonrodent animals, which seriously limits the advancement of stem cell biology and its ultimate translation to human clinical applications. Domestic cats are used frequently in biomedical research and are the preferred species for studies of normal physiology and disease, particularly in neuroscience. Therefore, the objective of this study was to characterize mesenchymal stem cells (MSC) from feline bone marrow for use in research on the application of stem cells to human health problems for which cats are the preferred model. METHODS Mesenchymal stem cells from feline bone marrow were isolated by standard methodology developed for other species and characterized according to morphology, growth traits, cell-surface antigen profile, and differentiation repertoire in vitro. RESULTS Feline mesenchymal stem cells exhibit a fibroblast-like morphology with bipolar or polygonal cell bodies and possess a cell-surface antigen profile similar to their rodent and human counterparts. Feline MSC exist at a frequency of 1 in 3.8 x 10(5) bone marrow mononuclear cells and are capable of differentiation to adipocytic, osteocytic, and neuronal phenotypes when exposed to appropriate induction media. CONCLUSIONS Mesenchymal stem cells isolated from feline bone marrow possess several traits typical of MSC from other species. Characterization of feline mesenchymal stem cells will facilitate future studies of stem cell biology and therapeutics for which the domestic cat is an indispensable model.

The prevalence of Aeromonas species in feces of horses with diarrhea.

Feces collected from 40 horses with diarrhea and 34 horses without diarrhea were examined to determine if an association existed between isolation of Aeromonas spp. and diarrhea. Samples were also examined for Salmonella spp., and identification of viruses and parasite ova. Neither Salmonella spp. nor Aeromonas spp. were isolated from the feces of 34 control horses. Aeromonas spp. were isolated from feces of 22 of 40 (55%) horses with diarrhea. Salmonella spp. were isolated from feces of 8 (20%) horses, and of these, 5 (12.5%) were also positive for Aeromonas spp. Twenty‐nine isolates of Aeromonas spp. were recovered from the feces of 22 diarrheic horses. Of these isolates, more than 80% were susceptible on in vitro testing to amikacin, ceftiofur, chloramphenicol, and gentamicin. All isolates were susceptible to enrofloxacin. Diarrheic horses positive for Aeromonas were significantly (P= .04) older than diarrheic horses negative for Aeromonas spp. A significantly greater number of fecal samples were positive for Aeromonas spp. during March through August than samples examined in other months (P= .014). Results of this study indicate that Aeromonas spp. should be considered as a cause of diarrhea in horses.

Experimental Transmission of Corynebacterium pseudotuberculosis Biovar equi in Horses by House Flies

Background The route of Corynebacterium pseudotuberculosis infection in horses remains undetermined, but transmission by insects is suspected. Objectives To investigate house flies (Musca domestica L.) as vectors of C. pseudotuberculosis transmission in horses. Animals Eight healthy, adult ponies. Methods Randomized, controlled, blinded prospective study. Ten wounds were created in the pectoral region where cages for flies were attached. Three ponies were directly inoculated with C. pseudotuberculosis. Four ponies were exposed for 24 hours to 20 hours C. pseudotuberculosis‐inoculated flies. One negative control pony was exposed to noninoculated flies. Ponies were examined daily for swelling, heat, pain, and drainage at the inoculation site. Blood was collected weekly for CBC and biochemical analysis, and twice weekly for synergistic hemolysis inhibition titers. Results Clinical signs of local infection and positive cultures were observed in 7/7 exposed ponies and were absent in the negative control. In exposed ponies, peak serologic titers (1 : 512 to 1 : 2,048) were obtained between days 17 and 21. Seroconversion was not observed in the negative control. Neutrophil counts were higher in the positive and fly‐exposed groups than in the negative control (P = .002 and P = .005) on day 3 postinoculation. Serum amyloid A concentrations were higher in the positive control than in the negative control and fly‐exposed ponies on days 3 (P < .0001) and 7 (P = .0004 and P = .0001). No differences were detected for other biochemical variables. Conclusions and Clinical Importance House flies can serve as mechanical vectors of C. pseudotuberculosis and can transmit the bacterium to ponies.

Presurgical antiseptic efficacy of chlorhexidine diacetate and providone-iodine in the canine preputial cavity.

Antiseptic flushing of the canine prepuce and its exclusion from the surgical field are recommended before abdominal surgery to reduce the risk of bacterial contamination. The authors cultured the preputial cavity of 60 dogs prior to and following flushing with 0.05% chlorhexidine diacetate, 1% povidone-iodine, or 0.9% saline control. Bacterial growth was evaluated using a semiquantitative method, and bacterial organisms were subsequently identified. There were no significant differences between povidone-iodine and the saline control in any of the variables assessed. Chlorhexidine resulted in a significant decrease in the proportion of positive postflush cultures compared with povidone-iodine. Although not significant, the difference in adverse reactions between povidone-iodine (25%) and chlorhexidine diacetate (5%) suggests clinical relevance. Based on the results of this study, a 2 min flush with 0.05% chlorhexidine diacetate is recommended for presurgical preparation of the preputial cavity.

Identification and characterization of mcr mediated colistin resistance in extraintestinal Escherichia coli from poultry and livestock in China

Antimicrobial resistance to colistin has emerged worldwide threatening the efficacy of one of the last-resort antimicrobials used for the treatment of multidrug-resistant Enterobacteriaceae infection in humans. In this study, we investigated the presence of colistin resistance genes (mcr-1, mcr-2, mcr-3) in Escherichia coli strains isolated from poultry and livestock collected between 2004 and 2012 in China. Furthermore, we studied the maintenance and transfer of the mcr-1 gene in E. coli after serial passages. Overall, 2.7% (17/624) of the E. coli isolates were positive for the mcr-1 gene while none were positive for the mcr-2 and mcr-3 genes. The prevalences of mcr-1 were similar in E. coli isolates from chickens (3.2%; 13/404), pigs (0.9%; 1/113) and ducks (6.8%; 3/44) but were absent in isolates from cattle (0/63). The mcr-1 gene was maintained in the E. coli after six passages (equivalent to 60 generations). In vitro transfer of mcr-1 was evident even without colistin selection. Our data indicate the presence of mcr-1 in extraintestinal E. coli from food-producing animals in China, and suggest that high numbers of the mcr-1-positive bacteria in poultry and livestock do not appear to be readily lost after withdrawal of colistin as a food additive.

Multidrug-resistant Escherichia coli and Tetracycline-resistant Enterococcus faecalis in Wild Raptors of Alabama and Georgia, USA

Abstract: Wild birds inhabit in a wide variety of environments and can travel great distances. Thus, wild birds can possibly spread antimicrobial resistance along the way, and this may represent a potential public health concern. We characterized antimicrobial resistance in fecal Escherichia coli and Enterococcus faecalis in wild raptors in the southeastern US. Cloacal samples were collected from 118 wild raptors of 17 species from 18 counties in Alabama and 15 counties in Georgia. A total of 112 E. coli and 76 E. faecalis isolates were recovered, and we found significantly more antimicrobial-resistant E. coli (20/112, 18%) than E. faecalis (6/76, 8%; P=0.05). Five antimicrobialresistant genes: blaTEM-1, blaCTX-M-1, tet(M), cmlA, cat, and gyrA, were identified in antimicrobial-resistant E. coli isolates. Five of 13 (38%) ampicillin-resistant E. coli harbored both bla-TEM-1 and blaCTX-M-1 genes, indicating they are extended-spectrum β-lactamase–carrying strains. Both of the tetracycline resistance genes, tet(M) and tet(L), were identified in E. faecalis isolates. Wild raptors seem to be a reservoir host of antimicrobial-resistant E. coli and E. faecalis and may represent a hazard to animal and human health by transmission of these isolates.

CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Passive protection against anthrax in mice with plasma derived from horses hyper-immunized against Bacillus anthracis Sterne strain

In this study, equine source polyclonal anti-Bacillus anthracis immunoglobulins were generated and utilized to demonstrate passive protection of mice in a lethal challenge assay. Four horses were hyper-immunized with B. anthracis Sterne strain for approximately one year. The geometric mean anti-PA titer in the horses at maximal response following immunization was 1:77,936 (Log2 mean titer 16.25, SEM ± 0.25 95% CI [15.5 –17.0]). The geometric mean neutralizing titer at maximal response was 1:128 (Log2 mean titer 7, SEM ± 0.0, 95% CI 7). Treatment with hyper-immune plasma or purified immunoglobulins was successful in passively protecting A/J mice from a lethal B. anthracis Sterne strain challenge. The treatment of mice with hyper-immune plasma at time 0 h and 24 h post-infection had no effect on survival, but did significantly increase mean time to death (p < 0.0001). Mice treated with purified immunoglobulins at time 0 h and 24 h post-infection in showed significant increase in survival rate (p < 0.001). Bacterial loads in lung, liver and spleen tissue were also assessed and were not significantly different in mice treated with hyper-immune plasma from placebo treated control mice. Mice treated with purified antibodies demonstrated mean colony forming units/gram tissue fourfold less than mice receiving placebo treatment (p < 0.0001). Immunotherapeutics harvested from horses immunized against B. anthracis Sterne strain represent a rapidly induced, inexpensive and effective expansion to the arsenal of treatments against anthrax.

Development of a sustained-release voriconazole-containing thermogel for subconjunctival injection in horses

Purpose To determine in vitro release profiles, transcorneal permeation, and ocular injection characteristics of a voriconazole-containing thermogel suitable for injection into the subconjunctival space (SCS). Methods In vitro release rate of voriconazole (0.3% and 1.5%) from poly (DL-lactide-co-glycolide-b-ethylene glycol-b-DL-lactide-co-glycolide) (PLGA-PEG-PLGA) thermogel was determined for 28 days. A Franz cell diffusion chamber was used to evaluate equine transcorneal and transscleral permeation of voriconazole (1.5% topical solution, 0.3% and 1.5% voriconazole-thermogel) for 24 hours. Antifungal activity of voriconazole released from the 1.5% voriconazole-thermogel was determined via the agar disk diffusion method. Ex vivo equine eyes were injected with liquid voriconazole-thermogel (4°C). Distension of the SCS was assessed ultrasonographically and macroscopically. SCS voriconazole-thermogel injections were performed in a horse 1 week and 2 hours before euthanasia and histopathologic analysis of ocular tissues performed. Results Voriconazole was released from the PLGA-PEG-PLGA thermogel for more than 21 days in all groups. Release followed first-order kinetics. Voriconazole diffused through the cornea and sclera in all groups. Permeation was greater through the sclerae than corneas. Voriconazole released from the 1.5% voriconazole-thermogel showed antifungal activity in vitro. Voriconazole-thermogel was easily able to be injected into the dorsal SCS where it formed a discrete gel deposit. Voriconazole-thermogel was easily injected in vivo and did not induce any adverse reactions. Conclusions Voriconazole-containing thermogels have potential application in treatment of keratomycosis. Further research is required to evaluate their performance in vivo.