Oral administration of a combination of select lactic acid bacteria strains to reduce the Salmonella invasion and inflammation of broiler chicks.

One-day-old chicks are susceptible to infection by strains of Salmonella enterica subspecies. Because multistrain probiotics are suggested to be more effective than monostrain probiotics due to the additive and synergistic effects, in this study, we prepared a multistrain formula A (MFA) consisting of 4 lactic acid bacteria (LAB) strains selected by enhancing the TNF-α production for mouse macrophage 264.7 cells. The antagonistic effect of this MFA against the cecal colonization, viscera invasiveness, as well as the inflammation of 1-d-old chicks challenged with Salmonella Typhimurium were then assayed. One-day-old chicks were fed with MFA from d 1 to d 3, and on d 4, chicks were challenged with Salmonella Typhimurium (200 µL, 10(6) cfu/mL). The livers, spleens, and cecal tonsils of chicks were then removed on d 3 and 6 postinfection. Compared with the multistrain formula B (MFB) which consisted of LAB strains selected at random, the efficacy of MFA to reduce the Salmonella counts recovered from the cecal tonsils, spleens, and livers of chicks were significantly higher. Moreover, when the levels of proinflammatory cytokines, such as IL-1ß, IL-6, interferon (IFN)-γ, and anti-inflmmatory cytokine, that is, IL-10, in cecal tonsils were measured by reverse-transcription real-time quantitative PCR; it was found that chicks fed with MFA for 3 d had lower levels of IL-1ß, IL-6, IFN-γ and a higher level of IL-10 in the cecal tonsils of chicks as compared with those of the chicks fed with MFB or without LAB. These results suggest that multistrain probiotics consisting of LAB strains selected by immunomodulatory activity and adherence are more effective than those consisting of strains selected at random in antagonistic effect against Salmonella colonization, invasion, and the induced inflammation.

Cellular assessment of crop lymphoid tissue from specific-pathogen-free white leghorn chickens after Salmonella enteritidis challenge.

The crop may be an important site along the upper alimentary tract in which a humoral immune response against Salmonella Enteritidis (SE) is elicited locally. The mucosal immune response within the crop (ingluvies) of specific-pathogen-free (SPF) white leghorn (WL) chickens against SE was investigated. Three trials were conducted using SPF WL pullets at age 5-6 wk. Trial 1 consisted of 77 birds evaluated for 10 wk post-SE infection (pi), trial 2 was composed of 72 birds monitored through 8 wk pi, and trial 3 was made up of 30 birds assessed for 5 wk pi. Birds were challenged per os with 10(8) colony-forming units/ml SE phage type 13. Crop lavage samples, crop tissues, ceca, and/or liver-spleen were collected preinfection and then at weekly intervals post-SE infection. Bacteriologic examination of cecal contents and/or liver-spleen occurred weekly to monitor progression of SE infection. Crop lavages were analyzed for SE-lipopolysaccharide (LPS)-specific immunoglobulin A (IgA) by enzyme-linked immunosorbent assay to assess humoral immune response. General histologic staining (hematoxylin and eosin [H&E] and methyl green-pyronin [MGP]) and immunohistochemical (IHC) staining (monoclonal antibodies CD45 and Bu-1) were applied to serial sections of crop to evaluate lymphoid tissue via light microscopy, to grade isolated lymphoid follicles (ILFs) by using score 0 (minimal, < 50 microm in diameter) to score 5 (sizable, > 200 microm in diameter) scale, and to characterize the cellular population of ILFs. Results revealed that cecum samples and liver-spleen samples were 100% SE culture positive at 1 wk pi, and then the percentage of SE positives progressively declined over time. Markedly increased crop SE-LPS-specific IgA antibodies were detected in crop samples by 2-3 wk pi, and the humoral response remained elevated above week 0 baseline for the duration of each trial. Crop ILFs of score 3 to 5 were observed in H&E-stained tissues, with an increased proportion of ILFs in post-SE-infected crops vs. uninfected. MGP staining showed plasma cells scattered within and at the periphery of ILFs. IHC staining revealed CD45 (pan-leukocyte) and Bu-1 (B-lymphocyte)-positive cells within crop ILFs. The chicken crop seems to be an organ in which lymphoid tissue may arise in response to enteric SE infection, and a site in which a humoral response may be generated against the SE pathogen.

Effect of organic acids in drinking water for young broilers on Campylobacter infection, volatile fatty acid production, gut microflora and histological cell changes.

Water is a prominent vehicle for Campylobacter spread throughout a chicken flock. The aim of this study was to evaluate the effect of organic acids administered through the drinking water, as a decontamination method, on gut microflora and the development of lesions in the gastrointestinal tracts of young broilers inoculated with 2 different doses of Campylobacter. The results revealed that most of the chickens were infected with Campylobacter at the end of the experiment. The drinking water was free of Campylobacter throughout the study. No difference of volatile fatty acid levels between treatment and control groups was observed in the crop and cecal contents. In the cecal contents, the total aerobic bacteria numbers were significantly higher in the treatment groups compared with the control groups (P < 0.01 and P < 0.04, respectively). Moreover, no damaged epithelial cells were observed in the chicken gut due to consumption of acidified drinking water. Acidified drinking water could therefore play a crucial role in a biosecurity strategy of preventing Campylobacter spread via drinking water in broiler flocks.

Response of the pigeon crop sac to mammotrophic hormones: comparison between relaxin and prolactin.

The effects of relaxin (RLX), a hormone that has previously been demonstrated to have mammotrophic properties, were studied in the pigeon crop sac, a well-known target organ for mammotrophic and lactogenic hormones, and compared with the effects produced by prolactin (PRL). The two hormones were injected directly over the crop at different doses and the response was evaluated after differing times of exposure. RLX causes a dose-related increase in wet and dry weights and [3H]thymidine and [3H]uridine uptake by the crop mucosa, as well as morphological changes indicating growth and differentiation of the epithelial cells similar to those occurring during physiological activation in incubation and hatching. At the doses assayed, the effects of RLX were nearly identical to those obtained following PRL in the short-term experiments, but differences in functional responses were found in the long-term experiment.

Pigeon crop-sac as a model system for studying the direct and indirect effects of hormones and growth factors on cell growth and differentiation in vivo.

The pigeon crop-sac is an underappreciated and underutilized model that can be used to study the direct and indirect effects of hormones, growth factors, and other agents on cell proliferation and differentiative processes in vivo. It can thus allow the uncertainties that attend many in vitro methods to be avoided. In addition, the crop mucosal cells are homogeneous and the organ is structurally much less complex than most other hormone-responsive target organs, such as the mammary gland or prostate. The organ is well suited for various other studies such as analysis of second-messenger systems for PRL and growth factors and the effects of growth-inhibitory substances.

Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli.

The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK2332 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1.5% of total cell protein.

Relaxin: a mammotropic hormone promoting growth and differentiation of the pigeon crop sac mucosa.

The pigeon crop sac is responsive to local administration of mammotropic hormones independently of systemic hormonal influences. We showed previously in mice that relaxin promotes growth of the mammary gland only after pre-treatment with estrogen. In this study we investigated whether relaxin, locally administered, has a stimulatory effect without estrogen pre-treatment. The results of this study show that relaxin stimulates the crop sac mucosa causing changes in its gross appearance and histological modifications indicating enhanced growth and differentiation of the mucosal epithelium. The effects of relaxin are dose-related, as demonstrated by the increases of the wet and dry weights, the [3H]thymidine and [3H]uridine uptakes of the mucosa, and the histochemically detectable DNA content of the mucosal epithelium. These results strengthen the idea that relaxin should be included in the list of mammotropic hormones.

Interactions among prolactin, epidermal growth factor, and proinsulin on the growth and morphology of the pigeon crop-sac mucosal epithelium in vivo.

The local pigeon crop-sac assay was used to test the direct effects of epidermal growth factor (EGF) and several other growth factors and hormones on the growth of mucosal epithelial cells in vivo. Insulin, relaxin, multiplication-stimulating activity, proinsulin, and platelet-derived or fibroblast growth factors had no direct stimulatory activity by themselves. Human insulin-like growth factor I and EGF caused dose-related stimulation, but were less effective than ovine (o) PRL. Insulin, platelet-derived growth factor, fibroblast growth factor, and multiplication-stimulating activity did not affect the proliferative response to oPRL when injected along with the hormone. Proinsulin augmented the direct mitogenic action of oPRL, but not that of EGF. When EGF was injected locally with PRL no interaction occurred even though both hormones were independently mitogenic. A single sc injection of a high dose of oPRL (0.5 mg) given at a site distant from the crop-sac had no effect on the mucosal epithelial cells, but it caused a significant increase in their response to the direct action of oPRL. However, the systemically acting PRL did not affect the direct local effect of EGF. The gross pattern of mucosal cell proliferation induced by PRL (parallel ridges resembling gastric rugae) differed from that produced by EGF (usually irregular patches), and PRL, but not EGF, promoted the accumulation of lipid droplets in the stimulated mucosal epithelial cells. Furthermore, the crop mucosal cells in the midline of the organ are unresponsive to PRL, but were highly responsive to EGF. These results indicate that although EGF and PRL are both mitogenic to crop mucosal epithelial cells, the former does not mimic the latter. They produce a different growth pattern, and EGF fails to promote differentiation of the resultant daughter cells. Moreover, EGF is a less specific mitogen than is PRL, and the two hormones do not interact in their mitogenic effects.

Regulation of cloned prolactin-inducible genes in pigeon crop.

Polyadenylated RNA from PRL-stimulated pigeon (Columba livia) crop was used as template to produce a cloned cDNA library in plasmids. The library was screened by differential hybridization against labeled nucleic acid populations representative of both unstimulated and PRL-stimulated crop tissue. By this method four independent clones coding for PRL-inducible mRNAs were identified. The regulation of these four genes ranged from modest (2- to 3-fold) to major (greater than 70-fold). A clone designated DA4 was complimentary to the most markedly stimulated crop mRNA. This mRNA encoded a polypeptide with a molecular weight of 35,500 which corresponds with the major induced protein synthesized in vivo. Messenger RNADA4 stimulation was dose dependent showing maximal induction by ovine PRL systemic injections in the 200 micrograms/day range. Above this dose PRL was less effective. The onset of mRNADA4 accumulation after a single PRL injection was rapid with statistically significant levels occurring by 3 h. Several lactogenic type hormones, but not an ungulate GH, were potent inducers of mRNADA4. The receptor responsible for mRNADA4 stimulation responds to mammalian lactogens (ovine PRL, human GH, human placental lactogen, bovine placental lactogen) and also can be blocked by an antibody to rabbit mammary gland PRL receptors. These results argue that regulation of pigeon crop gene expression (specifically mRNADA4 may be a relatively simple model of lactogenic hormone mechanisms.

The mitogenic, but not differentiative, response of crop tissue to prolactin is circadian phase dependent.

The circadian time of PRL administration is an important determinant of its stimulatory activity in pigeon crop tissue. Based on previously published experiments we chose two phases of the entrained circadian cycle (0 and 9 h after light onset) which represent minimum and maximum crop sensitivity and examined several specific biochemical markers of mitogenesis and differentiation. These included DNA synthesis, ornithine decarboxylase activity, total RNA concentration, polyadenylated RNA concentration, and a specific PRL-induced messenger RNA. In confirmation of previous studies, crop weight was increased twice as much by ovine PRL (0.5 micrograms/g BW X 3 days) injected 9 h after light onset compared with the 0 h time of injection. A single local injection of 10 micrograms of ovine-PRL increased DNA synthesis by 4-fold when injection was made at 9 h but had no effect when injection was made at 0 h after light onset. In contrast, PRL stimulation of gene expression, including total RNA, polyadenylated RNA, and a specific PRL-induced messenger RNA, were quantitatively identical at each phase of the circadian cycle. Corollary with its central role in cell proliferation, ornithine decarboxylase activity was induced by PRL injected at 9 h after light onset. The mitogenic and differentiational PRL effects in crop are therefore partially dissociable and may depend on distinct mechanisms.

The distribution of polypeptide YY (PYY) - and pancreatic polypeptide (PP) - immunoreactive cells in the domestic fowl.

The distribution of the polypeptide which has an N-terminal tyrosine and a C-terminal tyrosine (PYY) - and pancreatic polypeptide (PP) - immunoreactive cells were investigated in the gut of the domestic fowl. PPY-immunoreactive cells were observed in the duodenum and jejunum. PP-immunoreactive cells were seen in the duodenum, jejunum, ileum and colon. Both PYY- and PP-immunoreactive cells were extended from the basal lamina to the gut lumen i.e. of open type. PYY-immunoreactive cells occurred mainly in the basal and middle portion of the villi. On the other hand, PP-immunoreactive cells were located mostly in the crepts. The occurrence of PYY-immunoreactive cells in the upper part of the small intestine is rather similar to that of amphibians and reptiles, than to that of mammals, where PYY-immunoreactive cells are located in the distal part of the small intestine and in the large intestine.