Efficient production of chimeric mice from embryonic stem cells injected into 4- to 8-cell and blastocyst embryos

Background

Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem (ES) cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES cells into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator (PMM), and trace the fate of the injected ES cells.

Results

We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly.

Conclusions

These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.     

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.     

Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

Mouse in vivo-derived late 2-cell embryos have higher developmental competence after high osmolality vitrification and −80°C preservation than IVF or ICSI embryos

Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at −80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at −80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at −80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.     

Mitochondrial and DNA damage in bovine somatic cell nuclear transfer embryos

The generation of reactive oxygen species (ROS) and subsequent mitochondrial and DNA damage in bovine somatic cell nuclear transfer (SCNT) embryos were examined. Bovine enucleated oocytes were electrofused with donor cells and then activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. The H₂O₂ and ˙OH radical levels, mitochondrial morphology and membrane potential (ΔΨ), and DNA fragmentation of SCNT and in vitro fertilized (IVF) embryos at the zygote stage were analyzed. The H₂O₂ (35.6 ± 1.1 pixels/embryo) and ˙OH radical levels (44.6 ± 1.2 pixels/embryo) of SCNT embryos were significantly higher than those of IVF embryos (19.2 ± 1.5 and 23.8 ± 1.8 pixels/embryo, respectively, p < 0.05). The mitochondria morphology of SCNT embryos was diffused within the cytoplasm. The ΔΨ of SCNT embryos was significantly lower (p < 0.05) than that of IVF embryos (0.95 ± 0.04 vs. 1.21 ± 0.06, red/green). Moreover, the comet tail length of SCNT embryos was longer than that of IVF embryos (515.5 ± 26.4 µm vs. 425.6 ± 25.0 µm, p < 0.05). These results indicate that mitochondrial and DNA damage increased in bovine SCNT embryos, which may have been induced by increased ROS levels.     

Inhibition of cathepsin B activity reduces apoptosis by preventing cytochrome c release from mitochondria in porcine parthenotes

Cathepsin B, a lysosomal cysteine protease of the papain family, has recently been implicated in the quality and developmental competence of bovine preimplantation embryos. In this study, to determine whether inhibition of cathepsin B activity can improve porcine oocyte maturation and early embryo developmental competence, we supplemented in vitro maturation or embryo culture media with E-64, a cathepsin B inhibitor. Cathepsin B activity was high in poor quality germinal vesicle stage oocytes, but no differences in mRNA expression or protein localization were observed between good and poor quality oocytes, which were categorized based on morphology. Following treatment with 1 μM E-64, cathepsin B activity sharply decreased in 4-cell and blastocyst stage embryos. E-64 had no effect on cell number but significantly (P < 0.05) increased blastocyst formation and decreased the number of apoptotic cells in blastocysts. It also significantly (P < 0.05) enhanced mitochondrial membrane potential in blastocysts, reducing the release of cytochrome c and resulting in decreased expression of Caspase-3 and Caspase-9. In conclusion, inhibition of cathepsin B activity in porcine parthenotes using 1 μM E-64 resulted in attenuation of apoptosis via a reduction in the release of cytochrome c from mitochondria.     

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats

The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4–6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1⁺ cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4–6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.     

Fluoroquinolone Resistance Mechanism of Clinical Isolates and Selected Mutants of Pasteurella multocida from Bovine Respiratory Disease in China

The minimum inhibitory concentrations (MICs), mutation prevention concentrations (MPCs) and contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (ciprofloxacin, enrofloxacin and orbifloxacin) susceptibility in 23 Pasteurella multocida (Pm) isolates were investigated. Fluoroquinolone-susceptible isolates (MICs ≤0.25 µg/ml, 9 isolates) had no QRDR mutations, and their respective MPCs were low. Fluoroquinolone-intermediate isolates (MICs=0.5 µg/ml, 14 isolates) had QRDR mutations (Asp87 to Asn or Ala84 to Pro in gyrA), and their respective MPCs were high (4–32 µg/ml). First-step mutants (n=5) and laboratory-derived highly resistant fluoroquinolone mutants (n=5) also had QRDR mutations. The MICs of fluoroquinolones for mutant-derived strains were decreased in the presence of efflux inhibitors. The results indicated that the fluoroquinolone resistance of Pm is mainly due to multiple target gene mutations in gyrA and parC and the overexpression of efflux pump genes.     

Regulatory role of l-proline in fetal pig growth and intestinal epithelial cell proliferation

l-proline (Pro) is a precursor of ornithine, which is converted into polyamines via ornithine decarboxylase (ODC). Polyamines plays a key role in the proliferation of intestinal epithelial cells. The study investigated the effect of Pro on polyamine metabolism and cell proliferation on porcine enterocytes in vivo and in vitro. Twenty-four Huanjiang mini-pigs were randomly assigned into 1 of 3 groups and fed a basal diet that contained 0.77% alanine (Ala, iso-nitrogenous control), 1% Pro or 1% Pro + 0.0167% α-difluoromethylornithine (DFMO) from d 15 to 70 of gestation. The fetal body weight and number of fetuses per litter were determined, and the small and large intestines were obtained on d 70 ± 1.78 of gestation. The in vitro study was performed in intestinal porcine epithelial (IPEC-J2) cells cultured in Dulbecco''s modified Eagle medium-high glucose (DMEM-H) containing 0 μmol/L Pro, 400 μmol/L Pro, or 400 μmol/L Pro + 10 mmol/L DFMO for 4 d. The results showed that maternal dietary supplementation with 1% Pro increased fetal weight; the protein and DNA concentrations of the fetal small intestine; and mRNA levels for potassium voltage-gated channel, shaker-related subfamily, member 1 (Kv1.1) in the fetal small and large intestines (P < 0.05). Supplementing Pro to either gilts or IPEC-J2 cells increased ODC protein abundances and polyamine concentrations in the fetal intestines and IPEC-J2 cells (P < 0.05). In comparison with the Pro group, the combined administration of Pro and DFMO reduced the expression of ODC protein and spermine concentration in the fetal intestine, as well as the concentrations of putrescine, spermidine and spermine in IPEC-J2 cells (P < 0.05). Meanwhile, the percentage of cells in the S-phase and the mRNA levels of proto-oncogenes c-fos and c-myc were increased in response to Pro supplementation, whereas depletion of cellular polyamines with DFMO increased tumor protein p53 (p53) mRNA levels (P < 0.05). Taken together, dietary supplementation with Pro improved fetal pig growth and intestinal epithelial cell proliferation via enhancing polyamine synthesis.     

Laparoscopy vs. laparotomy for embryo transfer to produce transgenic goats (Capra hircus)

This study was performed to produce transgenic Korean native goat (Capra hircus) by laparoscopic embryo transfer (ET) to overcome the limitations of ET performed by laparotomy. Transgenic embryos were produced by DNA pronuclear microinjection of in vivo zygotes. The recipient goats were synchronized for estrus by using an introvaginal progesterone devices as a controlled internal drug-releasing insert (CIDR) for 13 days and injection of 400 IU PMSG 48 h before removal of the insert. Embryos were transferred on day 3 and 4 after removal of the insert. Recipient goats were deprived of feed for 48 h, then suspended in a laparotomy cradle at an angle of 45°. After obtaining a sufficient pneumoperitoneum, the laparoscope and forceps were inserted abdominally through 5 mm trocar sleeves. Examination of the ovaries and uterus was performed and then 213 embryos were transferred into the oviducts via the infundibula of 76 recipient goats. To compare pregnancy rates, ET was also performed by laparotomy in 82 recipient goats. The pregnancies in the recipient goats were diagnosed by ultrasound on day 30 after embryo transfer. The pregnancy rate with laparoscopic ET was significantly higher than with ET performed by laparotomy (46.1% vs. 28.6%, p < 0.05). In addition, the pregnancy rates were compared between ovulated and non-ovulated ovaries of the recipient goats in the laparoscopic ET group. No significant difference was observed between the pregnancy rates of ovulated and non-ovulated ovaries (41.3% vs. 33.3%, p < 0.05) suggesting that ET may also be possible in non-ovulated recipients through artificial rupture of Graafian follicles. These results suggest that laparoscopic ET is a highly efficient method for the transfer of goat embryos.     

RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.     

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T_m values of 85.03 ± 0.54℃ for stx1 and 87.47 ± 0.35℃ for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/µL), and quantifiable (R² = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.     

Dietary high protein-induced diarrhea and intestinal inflammation by activation of NF-κB signaling in piglets

The present study aimed to investigate whether inflammation-associated responses in piglets are induced by high protein (HP) through activating nuclear factor kappa B (NF-κB) signaling. Sixteen piglets (35 d of age, Duroc × [Landrace × Yorkshire], weaned at d 21, initial BW = 9.70 ± 0.11 kg) were allocated to 18% and 26% CP (HP group) at random, comprising 8 replicate pens per treatment. The piglets were slaughtered to collect intestinal tissues when apparent, persistent, and stable diarrhea syndromes happened (on d 12). No significant differences were observed in their growth performance (P > 0.05), but reduction by 19.11%, 25.31%, 23.64% of ADFI, ADG, and G:F, respectively was detected in the HP group. The HP group had greater (P = 0.002) diarrhea rates. Furthermore, dietary HP had lower ileal villus height (VH; P = 0.048), ratio of villus height to crypt depth (VH/CD ratio; P = 0.016), and colonic CD (P = 0.034), as well as had the trend (P = 0.075) to reduce the ileal villus absorptive area. Moreover, HP diets significantly elevated the goblet cell numbers in the ileal villi (P = 0.016) and colonic crypts (P < 0.001) and up-regulated (P = 0.012) the mRNA expression of mucin2 (Muc2) in the ileum. In addition, HP diets increased the myeloperoxidase concentration in the ileum (P = 0.002) and colon (P = 0.007) of piglets. Dietary HP significantly down-regulated the mRNA expression of tumor necrosis factor-α (TNF-α; P < 0.001) in the ileum, induced nitric oxide synthase (iNOS; P = 0.040) and interleukin-22 (IL-22; P = 0.008) in the colon, and inclined to down-regulate interleukin-1β (IL-1β; P = 0.076) expression in the colon. The relative protein abundance of Galectin-3 (P = 0.046) in the colon and the ratio of phosphorylation NF-κB to NF-κB (p–NF–κB/NF-κB ratio) in the ileum of HP piglets were also greater (P = 0.038). These results suggest that dietary HP may cause diarrhea in piglets by activating NF-κB signaling induced intestinal inflammation.     

Reproductive traits of the Ryukyu long-furred rat (Diplothrix legata) on Okinawa-jima Island

The Ryukyu long-furred rat, Diplothrix legata, is a large rodent distributed only on Amami-ohshima Island, Tokuno-shima Island and Okinawa-jima Island, Japan. This animal is endangered as a result of deforestation, predation by introduced carnivores and mortality caused by vehicles. We performed theriogenological examinations of 32 male and 25 female Ryukyu long-furred rats carcasses collected from wild populations on northern Okinawa-jima Island from December 2005 to September 2013. Adult males had remarkably large preputial glands. Seminiferous diameter of adult was significantly small (136 ± 28 µm, n=8) from April to August. Numerous spermatozoa were observed from September through February, and seminiferous diameter was significantly large (216 ± 27 µm, n=12) during this time in adults; testes length changed in a similar pattern. These findings indicate that the mating season may occur from September through February. Size (body length) at sexual maturity was estimated to be >560 mm in both sexes. From observation of corpora lutea and placental scars, litter size was estimated to range from 2 to 12 (average=6, n=4). These results provide fundamental knowledge that will be beneficial for in situ and ex situ conservation of this rare species.     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

The ability to enter into an avirulent viable but non-culturable (VBNC) form is widespread among Listeria monocytogenes isolates from salmon, patients and environment

Media-based bacteriological testing will fail to detect non-culturable organisms and the risk of consuming viable but non-culturable (VBNC) Listeria monocytogenes is unknown. We have here studied whether L. monocytogenes obtained from seafoods, processing environment and clinical cases enter the VBNC state and assessed the virulence of the non-culturable forms of the bacteria. A number of 16 L. monocytogenes strains were starved in microcosm water at 4 °C until loss of culturability. Metabolic activity in the VBNC form was measured as ATP generation using a luciferase assay and membrane integrity was examined using the LIVE/DEAD BacLight assay. All tested L. monocytogenes strains entered the VBNC state after starvation in microcosm water. Ongoing mRNA synthesis of hly in VBNC L. monocytogenes cells re-incubated in culture medium indicated a potential virulence of these forms. Sodium pyruvate and replenishment of nutrient were used in attempts to resuscitate VBNC cells. However, VBNC L. monocytogenes were not resuscitated under these conditions. VBNC L. monocytogenes were tested for virulence in a cell plaque assay and by intraperitoneally inoculation in immunodeficient RAG1^−/− mice. Inoculation of VBNC L. monocytogenes in immunodeficient mice did not cause morbidity, and plaque assay on HT-29 cells in culture indicated that the VBNC cells were avirulent. The results indicate that the risk of non-culturable L. monocytogenes in foods, when the VBNC state is induced by starvation, is negligible.     

Intraoperative Bleeding in Dogs from Grenada Seroreactive to Anaplasma platys and Ehrlichia canis

Background

Frequent exposure of Grenadian dogs to Rhipicephalus sanguineus results in Anaplasma platys, and Ehrlichia canis seroreactivity. During elective surgeries, substantial intraoperative hemorrhage occurs in some seroreactive dogs.

Objectives

To assess hemostatic parameters and bleeding tendencies as well as prevalence of PCR positivity in apparently healthy A. platys and E. canis seroreactive and seronegative free‐roaming dogs from Grenada.

Animals

Forty‐seven elective surgery dogs allocated to 4 groups: Seronegative control (n = 12), A. platys (n = 10), E. canis (n = 14) and A. platys, and E. canis (n = 11) seroreactive.

Methods

Preoperatively, hemostasis was assessed by platelet count, prothrombin time, activated partial thromboplastin time, and buccal mucosal bleeding time. Intra‐ and postoperative bleeding scores were subjectively assigned. Blood, spleen, bone marrow, and lymph node aspirates were tested by PCR.

Results

Bleeding scores in dogs coseroreactive for A. platys and E. canis were higher (P = .015) than those of seronegative dogs. A. platys DNA was amplified from 7/21 (33%) A. platys seroreactive dogs and from 1 E. canis seroreactive dog; E. canis DNA was amplified from 21/25 (84%) E. canis seroreactive dogs. E. canis DNA was amplified most often from blood, whereas A. platys DNA was amplified most often from bone marrow.

Conclusions and Clinical Importance

Apparently healthy, free‐roaming dogs coseropositive for A. platys and E. canis may have increased intraoperative bleeding tendencies despite normal hemostatic parameters. Future investigations should explore the potential for vascular injury as a cause for bleeding in these dogs. Improved tick control is needed for dogs in Grenada.     

Development of avian embryo manipulation techniques and their application to germ cell manipulation

The development of chicken embryo culture techniques, from single‐cell stage to hatching, makes it possible to manipulate developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blastodermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single‐cell stage, use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation of spermatozoa, and in vivo manipulation of gonads. So far, the only non‐viral method that has successfully produced transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cryopreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear transfer techniques for avian species is necessary.     

In vitro susceptibilities of Leptospira spp. and Borrelia burgdorferi isolates to amoxicillin, tilmicosin, and enrofloxacin

Antimicrobial susceptibility testing was conducted with 6 different spirochetal strains (4 strains of Leptospira spp. and 2 strains of Borrelia burgdorferi) against 3 antimicrobial agents, commonly used in equine and bovine practice. The ranges of MIC and MBC of amoxicillin against Leptospira spp. were 0.05-6.25 µg/ml and 6.25-25.0 µg/ml, respectively. And the ranges of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of amoxicillin against B. burgdorferi were 0.05-0.39 µg/ml and 0.20-0.78 µg/ml, respectively. The ranges of MIC and MBC of enrofloxacin against Leptospira spp. were 0.05-0.39 µg/ml and 0.05-0.39 µg/ml, respectively. Two strains of B. burgdorferi were resistant to enrofloxacin at the highest concentration tested for MBC (≥100 µg/ml). Therefore, the potential role of tilmicosin in the treatment of leptospirosis and borreliosis should be further evaluated in animal models to understand whether the in vivo studies will confirm in vitro results. All spirochetal isolates were inhibited (MIC) and were killed (MBC) by tilmicosin at concentrations below the limit of testing (≤0.01 µg/ml).