Monitoring of antimicrobial resistance in healthy dogs: first report of canine ampicillin-resistant Enterococcus faecium clonal complex 17

National surveillance programs on antimicrobial usage and antimicrobial resistance in animals have been established in various countries but few of them include bacteria from pets. The objectives of this study were to assess the prevalence of antimicrobial resistance in healthy dogs and to search for resistance phenotypes of clinical relevance. Escherichia coli and Enterococcus spp. were isolated from faecal swabs obtained from 127 dogs. Disk diffusion was used to measure antimicrobial susceptibility in 117 Escherichia coli, 10 Enterococcus faecium and 51 Enterococcus faecalis of canine origin. Resistance was relatively low compared with food animal species in Denmark. All Escherichia coli isolates were susceptible to broad-spectrum aminopenicillins, third generation cephalosporins and fluoroquinolones. Despite the low prevalence of resistance, statistical analysis of questionnaire data revealed a significant association (p=0.02) between recent antimicrobial treatment and resistance in Escherichia coli. Interestingly, two dogs were found to shed Enterococcus faecium resistant to ampicillin. Multilocus sequence typing of these isolates indicated that the two isolates belonged to sequence types associated with human nosocomial infections, and one (ST-192) was genetically related to human epidemic clonal complex 17. The detection of ampicillin-resistant Enterococcus faecium warrants further studies on the prevalence of these bacteria in dogs and on the possible implications to both animal and human health. The results suggest that distinct methods for detection and assessment of antimicrobial resistance in animals should be considered depending on the target animal species and the purposes of the study.     

A Case of Wound Botulism in a Foal Affected by Gastric Ulcers in Italy

The case of a 50-day-old Thoroughbred foal affected by sudden muscular weakness and failure to swallow associated with recumbency is described. The clinical picture suggested a diagnosis of botulism. At necropsy, nonglandular gastric mucosa presented deep ulcerations. No other macroscopic nor microscopic lesions were detected. Clostridium botulinum type B was isolated from the lesions of gastric mucosa, whereas the cecal and colonic content, assayed for both spores and toxins, were negative. Moreover, analyses of all potential environmental sources of toxins near the feeding areas were negative. It was concluded that the colonization of gastric ulcers may have contributed to this unusual case of botulism. To the best of our knowledge, this is the first case of botulism in horses reported in Italy, which is also the first case anywhere of wound botulism caused by infection of gastric mucosa lesions.     

A Single Sample Method for Evaluating 51Chromium-Ethylene Diaminic Tetraacetic Acid Clearance in Normal and Hyperthyroid Cats

Background: Chronic kidney failure is frequently seen in middle-aged and elderly cats. ⁵¹Chromium-ethylene diaminic tetraacetic acid (⁵¹Cr-EDTA) clearance and single blood sample (SBS) method are used in several species to estimate the glomerular filtration rate (GFR).
Hypothesis: The hypothesis of this study was that ⁵¹Cr-EDTA clearance could be determined using an SBS method in normal and hyperthyroid cats.
Animals: Forty-six cats were included in this study, with an average age of 9.5 years. Of these cats, 27 had hyperthyroidism; 19 were healthy.
Methods: After IV injection of ⁵¹Cr-EDTA (average dose: 4.25 MBq), 7 blood samples were obtained between 5 and 240 minutes. Reference clearance was calculated in mL/min and mL/min/kg body weight, using a 2-compartment model. Optimal time for clearance measurement with SBS was then determined by systematically comparing each individual plasma concentration to the reference multisample clearance.
Results: The average reference plasma clearance of ⁵¹Cr-EDTA for all cats was 14.9 mL/min (3.7 mL/min/kg). The clearance in hyperthyroid cats averaged 16.4 mL/min (4.3 mL/min/kg) and in normal cats averaged 10.3 mL/min (2.4 mL/min/kg).
The optimal time for the SBS was 48 minutes after injection of tracer ⁵¹Cr-EDTA ( R ²= 0.9414), giving the following converting equation: clearance = (0.0066 × DV_{48 minutes}) – 0.9277 (in mL/min).
Conclusions and Clinical Importance: In this study, the single sample ⁵¹Cr-EDTA clearance method was used to estimate the global GFR in cats. The method identified differences in clearance between normal and hyperthyroid cats. The optimal time for an SBS was 48 minutes.     

Testis-specific histone variants H2AL1/2 rapidly disappear from paternal heterochromatin after fertilization

Before fertilization, the genome packaging of male and female gametes is very different. Indeed, whereas the female haploid genome is associated with histones in a somatic-like chromatin structure, most of the male genome is tightly bound to protamines. However, it has recently been demonstrated that the pericentric heterochromatin regions of the male genome are associated with specific H2A-like histone variants, named H2AL1 and H2AL2, suggesting a heterogeneous organization. The fate and role of the sex-specific genome packaging transmitted by germinal cells to the embryo are not well understood. The aim of the present study was to follow reprogramming of the parental genomes in early embryos after in vivo fertilization. We show here that two typical epigenetic markers, trimethylated lysine 9 of histone H3 (TriMethylH3K9) and acetylated H4, are asymmetrically distributed between the parental genomes in one-cell mouse embryos, confirming data from embryos obtained after intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Indeed, whereas the maternal genome is highly enriched with trimethylH3K9, this mark is not detected in the paternal genome. On the contrary, histone H4 incorporated in the paternal genome is highly acetylated at an early stage, while in the maternal pronucleus, the level of acetylated H4 remains low in early one-cell embryos and becomes enriched at a later stage. Moreover, our results suggest a very quick disappearance of histone H2A variants H2AL1 and H2Al2 from the paternal pericentric heterochromatin regions after sperm-egg fusion.     

Cutaneous sterile pyogranuloma/granuloma syndrome in a dog

A case of canine cutaneous sterile pyogranuloma/granuloma syndrome (SPGS) with generalized asymmetrical alopecia and plaques is described. Results from histopathologic examination were suggestive of SPGS and failed to demonstrate any etiological agent. A systematic approach on how to definitively rule out infectious skin diseases is discussed.     

Physicochemical properties of meat of Italian Heavy Draft horses slaughtered at the age of eleven months

To investigate rheological and chemical characteristics of the meat from Italian Heavy Draft horse, 24 foals (12 males and 12 females) were weaned at 6 mo, reared and fed in the same way, and slaughtered at 11 mo of age. The results obtained showed that there were no significant differences between the sexes but that the muscle type is a significant variation source. The rectus femoris muscle was lighter, and the biceps femoris had a lower a* index than longissimus dorsii, rectus femoris, and semimembranosus muscle. The most tender muscle was the semitendinosus, and the toughest even after cooking was the biceps femoris. The male animals had a greater protein and lipid percentage. The acidic composition of the intramuscular fat showed a greater presence of MUFA (P < 0.05) in the females and of PUFA (P < 0.01) in the males. Colorimetry analysis of the subcutaneous and perirenal fat from these animals indicated the unfavorable yellow color seen in adult animals had not yet been acquired. The meat produced had low redness due to the low myoglobin content and high lightness. Besides, the low collagen content and its high solubility indicates an appreciable tenderness. The high level of unsaturation of the intramuscular fat resulted in a high ratio of unsaturated to saturated fat, making horse meat favorable from a health point of view.     

Effects of Ovum Pick-up Frequency and FSH Stimulation: A Retrospective Study on Seven Years of Beef Cattle In Vitro Embryo Production

The aim of this retrospective study was to compare the number of follicles, cumulus oocyte complexes (COCs) and cultured In Vitro Produced (IVP) embryos obtained from 1396 non‐stimulated Ovum Pick‐up (OPU) sessions on 81 donor animals in a twice weekly OPU scheme. Results were obtained from 640 sessions following FSH‐LH superstimulation, on 112 donors subjected to OPU once every 2 weeks. The stimulation protocol started with the insertion of an ear implant containing 3 mg norgestomet (Crestar, Intervet, Belgium) 8 days before puncture (day ?8). The dominant follicle was ablated by ultrasound‐guided follicle puncture on day ?6. On day ?3 and day ?2, cows were injected with FSH (Ovagen, ICP) twice daily (8 am to 8 pm ), i.e. a total dose of 160 μg FSH and 40 μg LG per donor per stimulation cycle. Animals were punctured 48 h after the last FSH injection (day 0). Progesterone implants were removed the next day. Stimulated donor cows were treated with this protocol at 14‐day intervals. Follicles were visualized with a Dynamic Imaging ultrasound scanner, equipped with a 6.5 MHz sectorial probe. Follicles were punctured with 55 cm long, 18 gauge needles at an aspiration pressure corresponding to a flow rate of 15 ml/min. Cumulus oocyte complexes were recovered and processed in a routine IVF set‐up. Results demonstrate that, expressed per session, FSH stimulation prior to OPU increases production efficiency with significantly more follicles punctured and oocytes retrieved. However, when overall results during comparable 2‐week periods are considered (four non‐stimulated sessions vs one stimulated), more follicles are punctured and more oocytes are retrieved using the non‐stimulated protocol. No significant differences in the number of cultured embryos could be detected, indicating that FSH/LH stimulation prior to OPU might have a positive effect on in vitro oocyte developmental competence as more embryos are cultured with less, presumably better‐quality, oocytes.     

Comparison of conservative management and juvenile pubic symphysiodesis in the early treatment of canine hip dysplasia.

OBJECTIVES: To evaluate the efficacy of juvenile pubic symphysiodesis (JPS) in a clinical setting for the early treatment of canine hip dysplasia (CHD), and to identify its indications and contraindications. METHODS: The final degree of CHD using the FCI (Fédération Cynologique Internationale) CHD classification in 5 Grades (A, B, C, D, E) was assessed at skeletal maturity in two homogeneous groups of dogs assessed at the age of 14 to 22 weeks and selected according to their susceptibility to CHD; one group was treated with JPS and one group was conservatively managed. Two hundred seventeen puppies completed the study; 81 were treated with JPS (group 1) and 76 were conservatively managed (group 2). A third group of 60 puppies with normal hips was followed as a negative control group. RESULTS: In group 1, 43.2% of the puppies had regression or a lack of progression of the disease in the final evaluation (Grade A & B), 25.9% had mild CHD (Grade C) and 30.9% had moderate and severe CHD (Grade D & E). In group 2, 23.6% of the puppies did not show any development of the disease (Grade A & B), 21.1% had mild CHD (Grade C) and 55.3% developed moderate to severe CHD (Grade D & E). Further investigation was done by comparing the severity of early signs of susceptibility to CHD with the final FCI Grades at adulthood in both groups. CLINICAL SIGNIFICANCE: The JPS procedure increased the odds of arresting or limiting the progression of CHD in mild to moderate grades of CHD, while it was less effective or ineffective in more severe forms.     

Identification and validation of housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in chickens

Real-time PCR has become a powerful tool for the detection of inflammatory parameters, including cytokines. Reference or housekeeping genes are used for the normalization of real-time RT-PCR results. In order to obtain reliable results, the stability of these housekeeping genes needs to be determined. In this study the stability of five genes, including beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phophoribosyl-transferase (HPRT), ubiquitin (UB) and glucose-6-phosphate dehydrogenase (G6PDH), was determined in a lipopolysaccharide inflammation model in chickens. beta-Actin appeared to be the most stable single gene in our model. Because the use of a single gene for normalization can lead to relatively large errors, the use of the geometric mean of multiple reference genes or normalization factor is preferred. The most stable combination for gene expression analysis in this lipopolysaccharide inflammation model in chickens is G6PDH and UB, since their correlation coefficients were 0.953 and 0.969, respectively (BestKeeper) and an M value of 0.34 and a low V(2/3) value of 0.155 (geNorm) were obtained. The use of HPRT and GAPDH should be avoided. The stable housekeeping genes, G6PDH and UB together, can be used to normalize the expression of pro-inflammatory cytokines in a lipopolysaccharide inflammation model in chickens.