Characterization of two unknown compounds in methanol extracts of rosemary oil

In this study, two unknown compounds in rosemary oil, containing 3% carnosic acid and 0.3% carnosol, were identified and characterized. After methanol extraction, purification, and analysis by reversed-phase HPLC and LC-MS, a recovery of 92% (+/-8%) of carnosic acid was obtained, but no carnosol was found. However, two unknown compounds with a molecular weight of 330.2 and 302.2 were consistently detected. From additional LC-MS-MS, (1)H NMR, and elemental analyses, it became clear that the first compound (M(w) = 330.2) could not be carnosol. It was hypothesized that it originated from the breakdown of the intramolecular bond of carnosol, followed by the addition of a water molecule. Possibly, an unsaturated double bond was formed after dehydration. Assuming that this compound was an intermediate in the conversion to rosmanol, the second unknown compound (M(w) = 302.2) may have resulted from the breakdown of the intramolecular bond of rosmanol. Similarly, an unsaturated double bond may have been formed. After splitting off carbon oxide, a detectable molecule with a molecular weight of 302.2 was observed.     

Osteomyelitis of the hyoid bones in two calves

Two calves were referred because of ptyalism and difficulty opening the mouth (Calf 1) and for elective umbilical hernia surgery under inhalation anaesthesia (Calf 2). Additional clinical signs were increased breath sounds and swelling in the region of the mandibular angle in Calf 1. Ultrasonography and endoscopy revealed oral inflammation and abscessation in the area of the base of the tongue in both calves. Infection of the hyoid apparatus was suspected based on ultrasonographic findings and confirmed by means of computed tomography. In Calf 1, there was no response to treatment with systemic antibiotics, nonsteroidal anti-inflammatory drugs and local lavage, and Calf 2 was not treated. Both calves were euthanized because of a poor prognosis and the diagnoses confirmed during postmortem examination. In Calf 1, the abscess was associated with complete destruction of the left epihyoid bone and partial destruction of the left stylohyoid and ceratohyoid bones. In Calf 2, the abscess was located at the distal end of the right stylohyoid bone near the epihyoid bone. Stomatitis or laryngeal and pharyngeal abscessation caused by sharp feed particles are common in cattle and infection of the hyoid apparatus should be included in the differential diagnosis.     

Faecal pH throughout the reproductive cycle of sows in commercial pig herds

Despite the importance of swine intestinal health, there are no easy-to-use and cost-efficient methods to evaluate it under field conditions for sows. To provide some indication about intestinal health, pH of faeces could be used but reference values for the currently high-performing commercial sow breeds are missing. In response, faecal pH of healthy sows from three different herds (herd A: 230 sows, herd B: 350 sows, herd C: 500 sows) was measured throughout the reproductive cycle. Within each herd, 10 sows were selected and rectal faeces samples were collected at different time points during the reproductive cycle: day 90 of gestation, day 1, 7, 14 and 21 of lactation, 7 days post-weaning and day 30 of the next gestation. In addition, data on sow body condition (back fat), feed composition and coarseness of the feed were collected. For individual pH measurements, the pH ranged from 6.30 to 7.93. However, for all herds together, the average faecal pH value of the sows throughout the reproductive cycle ranged from 6.89 to 7.15. Also, the variations due to sow and time of sampling during the reproductive cycle were low with coefficients of variation of less than 5%. The results from the pairwise comparisons of the statistical model showed that in the last stages of lactation (i.e., at day 21), significantly lower average pH values (p ≤ .05) are expected when compared to earlier stages of lactation (days 3 or 7), or at day 7 post-weaning or compared to day 30 of the next gestation. Bearing its limitations, our study provided reference faecal pH values from high-performing commercial sows under field conditions and as such they could be used directly in the field. Yet, further research is needed to provide more information on the factors affecting pH values throughout the reproductive cycle of the sow.     

Effect of vitamin E level and dietary zinc source on performance and intestinal health parameters in male broilers exposed to a temperature challenge in the finisher period

The objective of this study was to evaluate the interaction of zinc source (ZnSO₄ vs. zinc amino acid complex) and vitamin E level (50 IU vs. 100 IU) on performance and intestinal health of broilers exposed to a temperature challenge in the finisher period. A total of 1224 day old male Ross 308 broilers were randomly distributed among 4 dietary treatments (9 replicates per treatment). Dietary treatments were organized in a 2 × 2 factorial arrangement: two sources of zinc, 60 mg/kg of Zn as ZnSO₄.7H₂O or 60 mg/kg of Zn as zinc amino acid complexes (ZnAA) combined with two levels of vitamin E (50 or 100 IU/kg). Zinc and vitamin E were added to a wheat/rye-based diet that was designed to create a mild nutritional challenge. From day 28 until day 36 (finisher period), all birds were subjected to chronic cyclic high temperatures (32°C ± 2°C and RH 55–65% for 6 h daily). The combination of ZnAA and 50 IU/kg of vitamin E improved weight gain in the starter (day 0–10), finisher (day 28–36) and overall period (day 0–36) and feed conversion ratio in the starter (day 0–10) and finisher phase (day 28–36). Providing Zn as ZnAA significantly improved villus length and villus/crypt ratio in the starter, grower and finisher period and decreased infiltration of T-lymphocytes and ovotransferrin leakage in the finisher period. In conclusion, providing broilers with a diet supplemented with ZnAA and a vitamin E level of 50 IU/kg, resulted in better growth performance as compared to all other dietary treatments. Interestingly, under the conditions of this study, positive effects of ZnAA on performance did not occur when vitamin E was supplemented at 100 IU/kg in feed. Moreover, providing zinc as zinc amino acid complex improved intestinal health.     

Rapid testing and quantification of Salmonella in ileocaecal lymph nodes of Austrian pigs slaughtered for consumption

Traditionally, quantitative microbial risk assessment (QMRA) is based on culture-dependent technologies. However, molecular quantification could forge additional, detailed information. A prerequisite of quantitative real-time PCR in animal science is a tissue preparation method where large volumes of tissue material can be reduced and particularly target cells can be concentrated. An easy-to-use sample preparation method for food (Matrix-Lysis) was recently adapted to tissues and now permits quantification of target cells from up to 5 g of organic matrix. The aim of this study was to examine the suitability of Matrix-Lysis for quantification of Salmonella in porcine ileocaecal lymph nodes (ICLNs). After demonstrating constant recovery rates, ICLNs from 540 pigs were examined for Salmonella spp. with Matrix-Lysis. Samples were also analysed using ISO 6579:2002, a combined enrichment/qPCR method and a lateral flow test. It could be shown that qPCR coupled with Matrix-Lysis can contribute to QMRA in food safety by enabling reproducible quantitative data, even at low contamination rates.     

Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma

Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.     

Humoral and cell-mediated immune responses of d/d histocompatible pigs against classical swine fever (CSF) virus

A better understanding of cell-mediated immune responses to classical swine fever virus (CSFV) is essential for the future development of improved vaccines. We analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination. Viral infection induced high T cell responses with high primary and secondary CTL activity correlated with high IFN-gamma production, whereas vaccination with a live vaccine followed by infection mainly induced neutralizing antibody but low cell-mediated responses. Moreover, high IgG1 response was associated with high IFN-gamma response following infection whereas a weak IFN-gamma response was related to a good IgG2 response but a low IgG1 production. These data could reflect Th1/Th2-like balance of immune responses depending upon immunization protocols, which has not yet been described in the pig. T-cell responses to CSFV were evidenced by CSFV-specific CD25 upregulation on CD4-CD8+, but not on CD4+CD8- cells, which further illustrated the importance of CTL responses after infection. Our results indicated that generation of cell-mediated immune responses was much higher following intranasal/oral CSFV infection than after intramuscular vaccination, which implies that the capacity of new CSFV vaccines to induce higher T-cell responses should be considered.