Acute and long-term effects of antibiotics commonly used in laboratory animal medicine on the fecal microbiota.

Biomedical research relies on the use of animal models, and the animals used in those models receive medical care, including antibiotics for brief periods of time to treat conditions such as dermatitis, fight wounds, and suspected bacterial pathogens of unknown etiology. As many mouse model phenotypes are sensitive to changes in the gut microbiota, our goal was to examine the effect of antibiotics commonly administered to mice. Therefore, four treatment groups (subcutaneous enrofloxacin for 7 days, oral enrofloxacin for 14 days, oral trimethoprim-sulfamethoxazole for 14 days, and topical triple antibiotic ointment for 14 days) alongside a fifth control group receiving no treatment (n = 12/group) were included in our study. Fecal samples were collected prior to treatment, immediately after two weeks of exposure, and four weeks after cessation of treatment, and subjected to 16S rRNA library sequencing. The entire experimental design was replicated in mice from two different suppliers. As expected, several treatments including enrofloxacin and triple antibiotic ointment substantially decreased the amount of DNA recovered from fecal material, as well as the microbial richness. Notably, many of these effects were long-lasting with diminished gut microbiota (GM) richness four weeks following exposure, in both substrains of mice. Trimethoprim-sulfamethoxazole induced minimal to no discernible changes in the taxonomic composition beyond that seen in control mice. Collectively, these data highlight the need to consider the impact on GM of brief and seemingly routine use of antibiotics in the clinical care of research animals.

Effects of Fenbendazole-impregnated Feed and Topical Moxidectin during Quarantine on the Gut Microbiota of C57BL/6 Mice.

To protect the biosecurity of research rodent colonies, research institutions frequently require a quarantine period for live animals transferred into their facilities. Quarantine practices often include antibiotic and antiparasitic treatment with drugs such as fenbendazole and macrolide lactones. The influence of these compounds on the resident gut microbiota of mice is unknown, and any effects might subsequently affect model reproducibility. To test the influence of standard quarantine procedures on the composition of the microbiota, C57BL/6 mice, purchased from 2 different commercial suppliers, were randomly assigned to treatment groups (n = 12) by vendor and treated with fenbendazole-supplemented feed, topical moxidectin, both treatments, or no treatment (control), according to our institution's standard treatment regimen and duration. Feces were collected on arrival, immediately after completing the 8-wk treatment, and at 2 and 4 wk after treatment. Fecal DNA was extracted, sequenced, and analyzed to compare the changes in the microbiota of treated and control groups. Although significant main effects of time and treatment and interactions between those variables were detected in comparisons of richness, α-diversity, and ß-diversity, the effect sizes associated with any particular treatment were consistently much smaller than that associated with acclimation to a new facility in the absence of any quarantine treatments. This outcome, along with the visual evaluation of principal coordinate analysis based on multiple similarity indices, suggests that time or institution plays a larger role in alterations of the murine gut microbiota than do quarantine treatments on its composition.

Pharmacokinetics of Ceftiofur Crystalline-Free Acid in Clinically Healthy Dogs (Canis lupus familiaris).

Economical, injectable antibiotics are beneficial when clinical manifestations of an animal model prevent the use of oral antibiotics. Ceftiofur crystalline-free acid (CCFA) is an injectable, sustained-release form of ceftiofur, a third-generation cephalosporin that is labeled for use in swine, cattle, and horses. Because CCFA is an economical, injectable antibiotic that could be of value for use in research dogs, the objective of this study was to determine the pharmacokinetic properties of CCFA in apparently healthy dogs and to determine the minimal inhibitory concentrations of ceftiofur for veterinary pathogens cultured during 2011 through 2014 from the respiratory system, integumentary system, and urinary system of dogs. The study population comprised of 5 dogs (age, 1 y; weight, 24.7 to 26.9 kg) that were deemed healthy after no abnormalities were found on physical exam, CBC analysis, and clinical chemistry panel. Each dog received CCFA at 5.0 mg/kg SC, and blood samples were collected before administration of CCFA and at 1, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168, 192, 216, and 240 h after injection. The maximal plasma concentration (mean ± 1 SD) of CCFA was 1.98 ± 0.40 µ g/mL, time to reach maximal concentration was 22.3 ± 8.9 h, half-life was 56.6 ± 16.9 h, and AUC0-last was 124.98 ± 18.45 µ g-h/mL. The minimal inhibitory concentrations of ceftiofur ranged from ≤ 0.25 to ≥ 8.0 µ g/mL; ceftiofur was most effective against Pasteurella spp., Proteus spp., and Escherichia coli haemolytica and least effective against Bordatella bronchiseptica, Enterococcus spp., and Pseudomonas aeruginosa.

Transmission probabilities of mouse parvovirus 1 to sentinel mice chronically exposed to serial dilutions of contaminated bedding.

Intermittent serodetection of mouse parvovirus (MPV) infections in animal facilities occurs frequently when soiled bedding sentinel mouse monitoring systems are used. We evaluated induction of seroconversion in naïve single-caged weanling ICR mice (n = 10 per group) maintained on 5-fold serially diluted contaminated bedding obtained from SCID mice persistently shedding MPV1e. Soiled bedding from the infected SCID mice was collected, diluted, and redistributed weekly to cages housing ICR mice to represent chronic exposure to MPV at varying prevalence in a research colony. Sera was collected every other week for 12 wk and evaluated for reactivity to MPV nonstructural and capsid antigens by multiplex fluorescent immunoassay. Mice were euthanized after seroconversion, and DNA extracted from lymph node and spleen was evaluated by quantitative PCR. Cumulative incidence of MPV infection for each of the 7 soiled bedding dilution groups (range, 1:5 to 1:78125 [v/v]) was 100%, 100%, 90%, 20%, 70%, 60%, and 20%, respectively. Most seropositive mice (78%) converted within the first 2 to 3 wk of soiled bedding exposure, correlating to viral exposure when mice were 4 to 7 wk of age. Viral DNA was detected in lymphoid tissues collected from all mice that were seropositive to VP2 capsid antigen, whereas viral DNA was not detected in lymphoid tissue of seronegative mice. These data indicate seroconversion occurs consistently in young mice exposed to high doses of virus equivalent to fecal MPV loads observed in acutely infected mice, whereas seroconversion is inconsistent in mice chronically exposed to lower doses of virus.

Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer.

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (DeltaF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice.

Cloned transgenic swine via in vitro production and cryopreservation.

It has been notoriously difficult to successfully cryopreserve swine embryos, a task that has been even more difficult for in vitro-produced embryos. The first reproducible method of cryopreserving in vivo-produced swine embryos was after centrifugation and removal of the lipids. Here we report the adaptation of a similar process that permits the cryopreservation of in vitro-produced somatic cell nuclear transfer (SCNT) swine embryos. These embryos develop to the blastocyst stage and survive cryopreservation. Transfer of 163 cryopreserved SCNT embryos to two surrogates produced 10 piglets. Application of this technique may permit national and international movement of cloned transgenic swine embryos, storage until a suitable surrogate is available, or the long-term frozen storage of valuable genetics.

Generation of cloned transgenic pigs rich in omega-3 fatty acids.

Meat products are generally low in omega-3 (n-3) fatty acids, which are beneficial to human health. We describe the generation of cloned pigs that express a humanized Caenorhabditis elegans gene, fat-1, encoding an n-3 fatty acid desaturase. The hfat-1 transgenic pigs produce high levels of n-3 fatty acids from n-6 analogs, and their tissues have a significantly reduced ratio of n-6/n-3 fatty acids (P < 0.001).

Evaluation of the acute phase response in cloned pigs following a lipopolysaccharide challenge.

The objective of this study was to evaluate the acute phase response (APR) in cloned pigs derived from two different cell lines [C1 (n = 2) and C2 (n = 7)] as compared to genetically similar non-cloned pigs (CONT; n = 11) following a lipopolysaccharide (LPS; 25 microg/kg BW) challenge. Pigs were weaned at 21 days of age and maintained in individual pens in the same room until sample collection approximately 1 week later. Blood samples were collected every 30 min for 2 h prior to and 4h after the LPS challenge. Serum samples were analyzed for cortisol, tumor necrosis factor-alpha (TNF-alpha) and interleukin 6 (IL-6). Average gestational length for cloned pigs, 118.8 +/- 0.97 days, was longer (P < 0.005) than that of CONT pigs, 114+/-0.41 days. For serum cortisol, there was a time by group interaction (P < 0.0001) such that the cortisol response was greater in CONT pigs as compared to C2 pigs (P < 0.0001), but not different from C1 pigs (P > 0.74). A time by group interaction (P < 0.0001) was observed for serum TNF-alpha such that the TNF-alpha response was greater in CONT pigs as compared to C2 pigs (P = 0.0002) and tended to be greater (P < 0.06) than C1 pigs. A time by group interaction (P < 0.0001) was also observed for serum IL-6 such that the serum IL-6 response was greater (P < 0.003) in CONT pigs as compared to C2 pigs and there was a trend (P = 0.10) for serum IL-6 to be greater in CONT pigs compared to the C1 pigs. These are the first results to demonstrate that cortisol and proinflammatory cytokine profiles associated with the APR of cloned pigs are altered compared to genetically similar non-cloned pigs. Our results also indicate that the cell line from which clones are derived may dictate the APR. The hormone and cytokine profiles reported herein are a significant contribution towards our understanding, and perhaps our ability to prevent or reduce the incidence of premature deaths in cloned animals and warrants further investigation of the immune system of cloned animals.

Viral diseases of the rabbit.

Viral disease in the rabbit is encountered infrequently by the clinical practitioner; however, several viral diseases were reported to occur in this species. Viral diseases that are described in the rabbit primarily may affect the integument, gastrointestinal tract or, central nervous system or maybe multi-systemic in nature. Rabbit viral diseases range from oral papillomatosis, with benign clinical signs, to rabbit hemorrhagic disease and myxomatosis, which may result in significant clinical disease and mortality. The wild rabbit may serve as a reservoir for disease transmission for many of these viral agents. In general, treatment of viral disease in the rabbit is supportive in nature.