Competitive exclusion of Yersinia enterocolitica biotype 4, serotype O:3 by Yersinia enterocolitica biotype 1A,serotype O:6,30 in tissue culture and in pigs

AIMS: To study the adhesion properties of a biotype 4, serotype O:3 (human pathogenic) strain of Yersinia enterocolitica and to determine if adhesion in vitro and colonisation in vivo can be prevented by competition with a biotype 1A, serotype O:6,30 (non-pathogenic) strain. To study interaction between Y. enterocolitica biotype 4, serotype O:3 and cultured epithelial cells using the synthetic tripeptide arginine-glycine-aspartic acid (RGD).

METHODS: The human intestinal epithelial (HEp-2) cell line was used for in vitro studies. Inocula of Y. enterocolitica biotype 4, serotype O:3 radiolabelled using tritium were incubated with HEp-2 cells and RGD tripeptide, or with Y. enterocolitica biotype 1A, serotype O:6,30 sequentially or concurrently, then washed and lysed, and radioactivity measured to determine the effect of RGD on adhesion, and competitive exclusion of pathogenic by non-pathogenic bacteria. For in vivo studies, two groups of 5-week-old piglets (n=5/group) were sequentially inoculated orally with 5x10⁹ colony forming units (cfu) of either a non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica followed by a pathogenic biotype 4, serotype O:3 strain, or vice versa. Pigs were monitored for carriage of strains using bacterial culture and a multiplex polymerase chain reaction (PCR).

RESULTS: The RGD tripeptide significantly inhibited adherence of the pathogenic Y. enterocolitica strain to cultured epithelial cells, suggesting that adhesion involved the RGD tripeptide sequence. The non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica prevented adhesion of the pathogenic strain to cells in vitro when allowed to adhere first. Pathogenic Y. enterocolitica was consistently isolated from rectal swabs from 80-100% of pigs on all sampling occasions but not from oral swabs after 14 days in pigs first inoculated with the non- pathogenic strain or at 26 days in pigs first inoculated with the pathogenic strain.

CONCLUSIONS: A non-pathogenic strain of Y. enterocolitica reduced adhesion of a human pathogenic strain in vitro but not in vivo.     

A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

Post‐Warming Competence of In Vivo Matured Rabbit Oocytes Treated with Cytoskeletal Stabilization (Taxol) and Cytoskeletal Relaxant (Cytochalasin B) Before Vitrification

The aim of this study was to investigate the effect of Taxol and Cytochalasin B on the spindle, chromosome configuration and development to blastocyst stage after parthenogenesis activation of in vivo matured rabbit oocytes after vitrification. Oocytes were randomized into four groups: oocytes treated with Cytochalasin B or Taxol before vitrification, oocytes without treatment before vitrification and fresh oocytes. Oocytes were vitrified using Cryotop method, and meiotic spindle and chromosomal distribution were assessed with a confocal laser scanning microscopy. To determine oocyte competence, in vitro development of oocytes was assessed with parthenogenesis activation. There were no significant differences in the frequencies of normal spindle (33.0%, 31.0% and 32.6%, for non‐treated, Taxol‐treated and Cytochalasin B‐treated oocytes, respectively) and chromosome (48.3%, 46.6% and 34.8%, for non‐treated, Taxol‐treated oocytes and Cytochalasin B‐treated oocytes respectively) in vitrified groups, but significantly lower than those of fresh group (89.7% and 90.2%, for normal spindle and chromosome organization, respectively). No statistical differences were found in the cleavage and blastocyst development rates between non‐treated and Taxol‐treated oocytes (7.7% and 1.5% and 13.7% and 4.6%, for non‐treated and Taxol‐treated oocytes, respectively), although they were significantly lower than in the fresh group (42.3% and 32.1%, for cleavage and blastocyst development, respectively). Oocytes treated with Cytochalasin B failed to reach blastocyst stage. Normal spindle, chromosome configuration and blastocyst development of in vivo matured rabbit oocytes were damaged in vitrification, which was not improved by Taxol and Cytochalasin B pre‐treatment before vitrification. Moreover, a detrimental effect on blastocyst development of Cytochalasin B pre‐treatment before vitrification was observed.     

Comparison between S(+) ketamine–diazepam and S(+) ketamine–midazolam on anesthetic induction and recovery in dogs

S(+) ketamine, one of the two enantiomers of racemic ketamine, is a phencyclidine derivative that induces amnesia and analgesia. Its activity is related to blockade of NMDA receptors and some opioid action. We compared anesthetic induction and recovery quality with S(+) ketamine in combination with diazepam or midazolam in 10 dogs (ASA 1) admitted for elective surgery. After all clinical examinations, the dogs were separated into two groups (G I and G II). All animals received acepromazine (0.1 mg kg^?1) and fentanyl (5 µg kg^?1) IM, 20 minutes before induction with S(+) ketamine (6 mg kg^?1) and diazepam (0.5 mg kg^?1) IV (G I) or midazolam 0.2 mg kg^?1 (G II) IV. The doses of diazepam and midazolam were chosen according to the literature. All dogs were intubated and then maintained with halothane in oxygen at a vaporizer setting sufficient to maintain surgical anesthesia. Quality of induction, time needed for intubation, heart rate, respiratory rate, SpO₂, time to extubation, and quality of recovery were evaluated. The results were analyzed by Student's t‐test. Smooth induction and recovery were observed in all animals. The time to intubation was 45 ± 20 (GI) and 25 ± 6 seconds (GII), HR was 122 ± 12 (GI) and 125 ± 7 beats minute^?1 (GII), RR was 17 ± 2 (GI) and 21 ± 3 breaths minute^?1 (GII), SpO₂ was 96 ± 2 (GI) and 94 ± 1% (GII), time to extubation was 7 ± 3 (GI) and 4 ± 1 minutes (GII). No statistical differences were found in analyses, although time to intubation was less in GII. The results suggested that both combinations could be used safely for anesthetic induction in healthy dogs.     

Effects of Female Dietary Restriction in a Rabbit Growth Line During Rearing on Reproductive Performance and Embryo Quality

Maternal diet prior to mating has an effect on reproductive performance. We analysed the effect of maternal dietary restriction during rearing on reproductive performance, the embryo development and foetal growth. Females were categorized in two groups: (i) does with ad libitum access to feed or (ii) restricted. Two experiments were performed: (i) after 1 month, receptive females from both experimental groups were artificially inseminated and the reproductive performance was recorded during three reproductive cycles; at the first insemination, the body weight and perirenal fat thickness were recorded, and (ii) females from both experimental groups were inseminated, and 24 h later, embryos were recovered and transferred to recipient females from a maternal line. Later, embryonic implantation was assessed at day 14 by laparoscopy and foetal growth was monitored by ultrasound examination. In experiment 1, no differences in kindling rate was found, but prolificacy was showed to be higher in ad libitum does, which also were heavier than restricted ones. In experiment 2, no differences among does either in body weight, in perirenal fat thickness or in reproductive performance (ovulation rate and embryo recovery rate) were related to differences in feed intake. However, despite similar embryonic implantation losses, embryos from restricted females demonstrated higher foetal and gestational losses. Embryos from restricted does presented lower foetal growth than embryos from ad libitum does. Therefore, our results demonstrated that nutrition before first conception in a rabbit line selected for growth rate may impact on the embryo and results in a disturbance in gestational losses and foetal growth over all reproductive life.