A longitudinal study of Salmonella enterica infections in high-and low-seroprevalence finishing swine herds in The Netherlands.

The purpose of this investigation was to study the incidence and course of Salmonella infections in finishing pig herds in order to asses the stability of a given Salmonella herd status. Five low- and 7 high-seroprevalence herds were followed for seven sampling rounds. Each round, blood and faecal samples were tested in an indirect ELISA and by bacteriological culturing, respectively. In high-seroprevalence herds a positive Salmonella status was an indication of a long-term problem and the status was relatively stable over time. The herds experiencing clinical salmonellosis were not necessarily the herds with the highest seroprevalence. It is possible to deliver sero-negative finishers to the slaughterhouse, even though these pigs were seropositive as growers. In three out of five low-prevalence herds, major infection incidents occurred, indicating that changes in the Salmonella status should be anticipated. Low-prevalence herds can remain negative over a longer period of time as a result feeding a complete liquid feed containing fermented by-products.

Herd level husbandry factors associated with the serological Salmonella prevalence in finishing pig herds in The Netherlands.

A national program to reduce Salmonella in pork and pork products should include monitoring and intervention at farm level. To develop an adequate intervention strategy at farm level, risk factors for Salmonella infections in finishing pigs have to be determined. In this study, blood samples were collected randomly at two slaughterhouses from slaughter pigs. Samples were tested by the Dutch Salmonella ELISA, based on the O-antigens 1, 4, 5, 6, 7 and 12, using a cut-off of OD%=10. This ELISA has been calibrated against the Danish ELISA to give comparable results. Workers from herds from which at least forty blood samples had been collected, were asked to participate in a questionnaire. In total, 353 questionnaires were obtained and analysed. Significant risk factors associated with the proportion of seropositive samples were identified by multiple linear logistic regression. The feeding of a complete liquid feed containing fermented by-products and the omission of disinfection after pressure washing a compartment as part of an all-in/all-out procedure, were both associated with a lower Salmonella seroprevalence. A small to moderate herd size (<800 finishing pigs), a previous diagnosis of clinical Salmonella infection in the herd, the use of tylosin as an antimicrobial growth promoter in finishing feed, or herds which had more than 16% of the livers of their pigs condemned at the slaughterhouse as a result of white spots were associated with a higher Salmonella seroprevalence. Hypothetical intervention strategies based on these risk factors can be studied for their effect on the Salmonella seroprevalence and practical applicability in field studies.

Gonadotropin-releasing hormone receptor activation of extracellular signal-regulated kinase and tyrosine kinases in transfected GH3 cells and in alphaT3-1 cells.

GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.

Effect of CRH on the preovulatory LH and FSH surge in the cyclic rat: a role for arginine vasopressin?

The effect of intracerebroventricular (i.c.v.) injection or infusion of various doses of corticotropin-releasing hormone (CRH) on the LH and FSH surge was studied in pro-oestrous rats supplied with a jugular vein and an i.c.v. cannula. Additionally, we investigated if arginine vasopressin (AVP) was involved in the CRH-induced alterations to the surge of gonadotropins. I.c.v. injection of 10 micrograms CRH given 5 min before the presumed onset of the LH surge caused a strong inhibition of the LH surge and a slight inhibition of the FSH surge. Three to four h after CRH injection, its inhibitory effect diminished. A 6'h i.c.v. infusion of CRH started 1 h before the presumed onset of the LH surge, caused a dose-related inhibition of the LH and FSH surge. Infusion of 1 micrograms/h CRH did not suppress the surge of both hormones while infusion of 5 or 10 micrograms/h CRH inhibited the LH surge. Infusion of 10 micrograms/h CRH caused a strong suppression of plasma LH during the first 3 h of the LH surge. Despite continuation of CRH infusion, the inhibitory effect disappeared and plasma LH increased to similar levels as in controls at corresponding points of time of the LH surge. The FSH surge was also suppressed by infusion of 10 micrograms/h CRH. The surge of LH and FSH was not affected by a 9-h infusion of 10 micrograms/h CRH started 4 h before the presumed onset of the LH surge. This observation also indicates that the inhibitory effect of CRH may last for only 3-4 h. The surge of LH and FSH was not affected by i.c.v. injections of AVP-antiserum. However, pretreatment with AVP-antiserum prolonged the inhibitory effect of CRH on the LH surge. In conclusion, CRH can inhibit the pro-oestrous LH and to a lesser extent the FSH rise for only 3-4 h after the beginning of CRH administration. AVP may play a role in limiting the inhibitory effect of CRH on LH to 3-4 h.

Activation of MAP kinase by the LHRH receptor through a dual mechanism involving protein kinase C and a pertussis toxin-sensitive G protein.

The LHRH receptor in alpha T3-1 gonadotrope cells was shown to bring about a marked and sustained activation of MAP kinase. This response was prevented by protein kinase C inhibition or down-regulation and could be partially mimicked by phorbol ester. Additional evidence for inhibition of this response by pertussis toxin and partial mimicry by mastoparan (in a pertussis toxin-sensitive manner) provides the first evidence for Gi/Go-mediated signal transduction by the LHRH receptor. This dual mechanism of MAP kinase activation appears to be exceptional amongst the G protein-linked receptors that have been investigated.

Effect of tyrosine kinase inhibitors on luteinizing hormone-releasing hormone (LHRH)-induced gonadotropin release from the anterior pituitary.

A range of selective tyrosine kinase inhibitors, piceatannol, methyl-2,5-dihydroxycinnamate (MDC), genistein, psi-tectorigenin and lavendustin A, all reduced luteinizing hormone-releasing hormone (LHRH)-induced luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from pro-oestrous rat hemipituitaries incubated in vitro. In general, both 'initial' release and the augmented release resulting from LHRH self-priming, were reduced in parallel in a concentration-dependent fashion. The effects of piceatannol were independent of the steroidal status of the pituitary tissue. Both piceatannol and MDC greatly reduced LH release by ionomycin and a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), suggesting that the tyrosine kinase(s)-dependent step is in the later stages of the stimulus-secretion pathway activated by the LHRH receptor. These data were supported by immunoblots for phosphotyrosine showing that in the gonadotrope-derived alpha T3-1 cell line, treatment with LHRH caused piceatannol-sensitive increases in specific tyrosine phosphorylation of several proteins (major bands at 65-75 and 120-130 kDa). Treatment of cells with PDBu mimicked the tyrosine phosphorylations evoked by LHRH whereas the PKC inhibitor, GF109203X, partially reduced both LHRH- and PDBu-induced tyrosine phosphorylations. Direct effects of MDC and piceatannol on PKC were assessed in an in vitro PKC assay; piceatannol, but not MDC, inhibited PKC activity but at considerably higher concentrations than required for inhibition of LHRH-induced gonadotropin secretion. These data support a role for tyrosine kinase activation in LHRH-induced secretion.

The differential effects of protein kinase C activators and inhibitors on rat anterior pituitary hormone release.

We investigated the possibility that various protein kinase C (PKC) activators and inhibitors may differentially affect luteinizing hormone (LH) and growth hormone (GH) release from rat anterior pituitary tissue, incubated in vitro. Activators of PKC induced LH release with the following order of potency: mezerein > phorbol 12,13-dibutyrate (PDBu). Mezerein and PDBu were equipotent on GH release. A range of PKC inhibitors (including compounds highly selective for PKC) potently and completely inhibited PKC activator-induced LH and GH release. Chelerythrine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) were less potent inhibitors of PDBu-induced GH release than of LH release. A component of PDBu- and mezerein-induced LH release was inhibited by H7 with high potency, but a second H7-insensitive component was detected. Mezerein- and PDBu-induced GH release consisted of an H7-resistant component only. When the regulatory domain of PKCs from different sources was investigated by displacement of [3H]PDBu binding, the affinity for mezerein was 3-5-fold greater than that for PDBu at PKCs from cerebral cortex, lung and alpha and beta isoforms extensively purified from brain. Anterior pituitary PKCs were unusual in showing closely matched affinity for mezerein and PDBu, reminiscent of their equivalent potency on GH release. In order to investigate the potency of the catalytic domain inhibitor H7 on PKCs from different sources, enzyme activity assays were carried out on partially purified cytosolic PKCs from midbrain and anterior pituitary and on extensively purified PKC alpha and PKC beta. The Ca(2+)-independent component of PDBu-induced (phosphatidylserine-dependent) activity from anterior pituitary alone showed unusually low potency of inhibition by H7 but was potently inhibited by staurosporine and Ro 31-8220. In contrast, the Ca(2+)-dependent PKC activity in anterior pituitary was inhibited by H7, staurosporine and Ro-31-8220 with high potency as in all other preparations. These results are consistent with the presence and active role in secretion of pharmacologically distinct forms of PKC (or PKC-like kinases) in rat anterior pituitary cells.