Purified beta-glucan as an abiotic feed additive up-regulates the innate immune response in immature chickens against Salmonella enterica serovar Enteritidis.

Functionally, the innate immune system of immature chickens is inefficient during the first week posthatch. This immunological inefficiency enables pathogens such as Salmonella enterica serovar Enteritidis (SE) to invade and colonize the visceral organs of immature chickens. The objective of this study was to evaluate the effect of purified beta-glucan as an immunomodulator of the innate immune response. beta-glucan, as a feed additive, significantly provided protection against SE organ invasion in young chickens (P<0.05). The functional efficiency of heterophils isolated from neonatal chickens fed a beta-glucan ration was significantly (P<0.05) up-regulated when compared to heterophils isolated from chickens fed a control ration as determined with an array of functional assays. Phagocytosis, bactericidal killing, and oxidative burst were significantly increased in heterophils isolated from chickens fed the purified beta-glucan ration (P<0.05). To our knowledge, this is the first report of a purified beta-glucan feed additive significantly decreasing the incidence of SE organ invasion in immature chickens and up-regulating the functional abilities of heterophils isolated from immature chickens against an invading pathogen, SE.

Age-dependent phagocytosis and bactericidal activities of the chicken heterophil.

Chicks are most susceptible to Salmonella infection during the first 4 days post-hatch. In poultry, one of the primary cells in the innate immune response to early bacterial invasion by Salmonella is the heterophil. Previous studies using a granulocytopenic chicken model in more mature birds demonstrated the significant role heterophils have in the defense mechanism against Salmonella. In the past studies have also shown the efficiency of heterophils from 3- to 5-week-old chickens to phagocytose and kill Salmonella as compared to monocytes. During the present study, we investigated the phagocytic and bactericidal activities of heterophils from chickens during the first 7 days post-hatch to evaluate whether decreased heterophil function plays a role in the susceptibility of young chicks to Salmonella infections. Peripheral blood counts demonstrated no differences in the percentages of heterophils during the first week post-hatch. The phagocytic index of the heterophil did not change on day 1 or day 4, but doubled by day 7 (day 1, 30.69; day 4, 33.99; day 7, 60.46). Interestingly, the bactericidal activity of the heterophils from all three age groups efficiently killed Salmonella enteritidis. Based on this data, we conclude that a relationship exists between the age of the chick, the functional activity of the heterophil, and the susceptibility to organ invasion by Salmonella.

Ontogeny of the phagocytic and bactericidal activities of turkey heterophils and their potentiation by Salmonella enteritidis-immune lymphokines.

Heterophils, the functional equivalent to the mammalian neutrophil, are important mediators of natural resistance against invasive pathogens in poultry. Young poultry are susceptible to pathogens, such as Salmonella enteritidis, during the first week post-hatch. No studies have evaluated the ontogeny of heterophil function in turkeys during the first few weeks post-hatch. Previous studies from our laboratory have shown day-old poults were protected against S. enteritidis organ invasion following immunoprophylactic administration of chicken S. enteritidis immune lymphokines. Therefore, the objective in the present study was to characterize the development of phagocytosis and bacterial killing by turkey heterophils during the first 3 weeks of life and to compare the effect of immune lymphokines on the development of heterophil phagocytosis and killing during the first 3 weeks post-hatch. Both functional phagocytosis and killing activities were age-dependent events. During the first 1-7 days post-hatch, little functional activity was demonstrated which apparently is associated with susceptibility. Optimal heterophil phagocytosis and killing activities were reached 14-21 days post-hatch. Administration immune lymphokines significantly potentiated phagocytosis (P < 0.01) and killing (P < 0.001) by turkey heterophils. In fact, immune lymphokine administration to 1-7-day-old poults augmented phagocytosis and killing activities of heterophils equivalent to levels found in functionally mature 14-21-day-old poults. These results demonstrate the ontogeny of the functional activity of the turkey heterophil is an age-related phenomenon, with inefficient phagocytosis and killing during the first week post-hatch. Prophylactic administration of immune lymphokines significantly potentiated the functional activity of the heterophil poults during the first 3 weeks of life. Most importantly the administration of immune lymphokines enhanced the functional activity of heterophils from 1-7-day-old poults to levels comparable to that of an immunologically mature bird.

Efficacy of Salmonella enteritidis-immune lymphokines on horizontal transmission of S. arizonae in turkeys and S. gallinarum in chickens.

Salmonella arizonae (SA) and S. gallinarum (SG) are of economic importance to international poultry production because of their pathogenesis in young poultry during the first week after hatching. Previous studies from our laboratory have shown immune lymphokines (ILK) produced by S. enteritidis (SE)-immunized chickens provide protection against SE organ invasion in day-old chickens and turkey poults. Previous studies have also demonstrated that SG organ invasion was significantly decreased by administration of ILK to broiler chicks. The objective of the present study was to determine the effect of ILK on the incidence of horizontal transmission of SA in turkey poults and of SG in broiler chicks. The effect of ILK administration on horizontal transmission of SA in poults and SG in chicks was assessed in a seeder/contact model. Seeders were challenged with the appropriate bacterium (SA turkeys, SG chicks), contacts were either untreated or administered ILK. Seeders and contacts cohabited within an experimental group throughout the experiment. Mortality and organ invasion as a result of horizontal transmission were determined. There were no significant differences in mortality between non-treated and ILK-treated contact poults. In contrast, SG was extremely pathogenic to young broiler chicks. Non-treated contact chicks had a mortality rate of approximately 68% whereas significant (P < 0.05) reduction in mortality was demonstrated in the contact chicks treated with ILK (15%). Horizontal transmission, as determined by organ invasion, of SA to contact turkey poults and SG to contact broiler chicks was also significantly (P <0.05) decreased by immunoprophylactic administration of ILK. Bacterial recovery of SA from the liver/spleen and the cecal tonsil in contact poults and SG from contact chicks treated with ILK was dramatically reduced when compared to non-treated contact poults and chicks. Our results strongly suggest the immunoprophylactic administration of SE-immune lymphokines to young turkey poults and broiler chicks significantly reduces the horizontal transmission of Salmonella in poultry. These results suggest the possibilities of using a non-vaccine immunologically-based preventative strategy against Salmonella in poultry.

Enhancement of phagocytosis and bacterial killing by heterophils from neonatal chicks after administration of Salmonella enteritidis-immune lymphokines.

During the first week post-hatch, chickens demonstrate an increased susceptibility to infection by bacteria such as Salmonella. The purpose of the present study was to evaluate the effects of immune lymphokines on phagocytosis and killing activities of heterophils in chicks during the first 1-7 days of life. Lymphokines isolated from chicken splenic T-cells harvested from Salmonella enteriditis (SE)-hyperimmunized hens (SE-ILK), have in past experiments, demonstrated augmentation of heterophil activity in day-of-hatch chicks resulting in protection from SE organ invasion. The present experiments reveal significant increases (p<0.05) in heterophil phagocytosis and killing when comparing chicks treated with SE-ILK to control groups in vitro. In SE-ILK-treated groups, a two-fold or greater increase is noted in heterophil phagocytosis within I h of incubation as compared to controls. Heterophils isolated from 1-day-old and 4-day-old chicks treated with SE-ILK killed significantly greater numbers (p<0.05) of SE than heterophils isolated from control groups. By Day 7 post-hatch, significance is not noted in the killing activity of heterophils from treated groups when compared to control groups. However, heterophils from SE-ILK groups continue to kill greater numbers of SE than control groups. These data support SE-ILK augmentation results in an enhanced heterophil function in chicks during the greatest period of susceptibility to Salmonella invasion.

Differential activation of signal transduction pathways mediating phagocytosis, oxidative burst, and degranulation by chicken heterophils in response to stimulation with opsonized Salmonella enteritidis.

The activation of signal transduction pathways is required for the expression of functional enhancement of cellular activities. In the present studies, initial attempts were made to identify the signal transduction factors involved in activating phagocytosis, generation of an oxidative burst, and degranulation by heterophils isolated from neonatal chickens in response to opsonized Salmonella enteritidis (opsonized SE). Peripheral blood heterophils were isolated and exposed to known inhibitors of signal transduction pathways for either 20 min (staurosporin, genistein, or verapamil) or 120 min (pertussis toxin) at 39 degrees C. The cells were then stimulated for 30 min at 39 degrees C with opsonized SE. Phagocytosis, luminol-dependent chemoluminescence (LDCL), and beta-D glucuronidase release were then evaluated in vitro. The G-protein inhibitor pertussin toxin markedly inhibited (>80%) phagocytosis of opsonized SE. Both the protein kinase inhibitor (staurosporin) and calcium channel inhibitor (verapamil) reduced phagocytosis in a dose response manner. Genistein, a tyrosine kinase inhibitor, had no effect on phagocytosis. Staurosporin had a marked inhibitory effect on LDCL (>90%) while genistein had a dose responsive inhibition on LDCL. Both verapamil (40-45%) and pertussin toxin (50-55%) had a statistically significant, but less biologically significant effect on LDCL. Genistein significantly reduced the degranulation (78-81%) of heterophils by opsonized SE. Staurosporin also reduced degranulation by 43-50%, but neither verapamil nor pertussis toxin had a significant effect on degranulation. These findings demonstrate that distinct signal transduction pathways differentially regulate the stimulation of the functional activities of avian heterophils. Pertussin toxin-sensitive, Ca++-dependent G-proteins appear to regulate phagocytosis of opsonized SE, protein kinase C-dependent, tyrosine kinase-dependent protein phosphorylation plays a major role in LDCL, and tyrosine kinase(s)-dependent phosphorylation regulates primary granule release.

Resistance to Salmonella enteritidis organ invasion in day-old turkeys and chickens by transformed T-cell line-produced lymphokines.

We previously reported an increased resistance to Salmonella enteritidis (SE) organ invasion in chicks and turkey poults injected prophylactically with SE-immune lymphokines (ILK). In the present study, concanavalin A (Con-A)-activated splenic T cells isolated from SE-hyperimmunized hens were transformed in vitro with reticuloendotheliosis virus strain T (REV-T) (chicken syncitial virus). These transformed T cells were then maintained as a long-term (> 1 yr) cell line for the harvest of immune lymphokines (VILK). The efficacy of VILK to protect turkey poults and chicks against SE organ invasion and the correlation between organ invasion and peripheral blood heterophilia were then evaluated. Three groups of day-old poults and chicks were injected intraperitoneally with either phosphate-buffered saline (PBS; group A), ILK (group B), or VILK (group C). Thirty minutes postinjection, poults and chicks were challenged per os with 5 x 10(5) colony-forming units (CFU) SE and 5 x 10(4) CFU SE, respectively. At 24 hr posttreatment, birds in groups A, B, and C were euthanatized and liver samples were cultured for the presence of SE. Both the VILK- and ILK-treated turkeys and chicks had significant reductions in organ invasion when compared with the PBS-injected controls (P < 0.005). For peripheral blood studies, turkeys and chicks were treated as above, and at 4 hr post-PBS, ILK, or VILK injection; total and differential peripheral blood counts were performed on birds from each group. A significant (P < 0.05) peripheral blood heterophilia at 4 hr postinjection was observed in the ILK- and VILK-treated birds, with no such increase found in the PBS-injected group. Correlation analysis revealed a direct relationship between the peripheral blood heterophilia in turkeys and chicks seen at 4 hr postinjection and the reduction in SE organ invasion seen in the VILK and ILK treatment groups (r = 0.991, r = 0.91, respectively). T cells isolated and transformed from nonimmune chickens did not produce factors that protected chicks from SE organ invasion and did not cause the peripheral blood heterophilia observed with ILK and VILK. These results show that the virally transformed SE-immune T-cell line produces lymphokines that result in the same level of peripheral blood heterophilia as ILK and was equally protective against SE organ invasion as ILK.

Effect of a commercial competitive exclusion culture (Preempt) on mortality and horizontal transmission of Salmonella gallinarum in broiler chickens.

A commercial competitive exclusion (CE) culture (PREEMPT) was evaluated for its ability to decrease mortality during the first 10-12 days posthatch resulting from the causative agent of fowl typhoid, Salmonella gallinarum. In the first experiment, chicks provided the CE culture on day of hatch and challenged with 10(5) S. gallinarum on day 3 had a significant decrease in mortality compared with non-CE-treated S. gallinarum-challenged chicks in all four replicates. Mortality for control chicks when averaged across all four replicates was 74% compared with 7.5% for the CE-treated chicks. A second experiment was performed that was designed to measure the efficacy of the CE culture in decreasing the horizontal transmission of this pathogen from infected to uninfected chicks when commingled. Day-of-hatch chicks that were directly infected (seeders) with 10(5) S. gallinarum and provided no CE culture averaged 86% S. gallinarum organ positive and 80% mortality during the first 12 days posthatch across four replicates. Untreated contact (not directly infected) chicks that were commingled with the seeder chicks averaged 84% S. gallinarum organ positive and 54% mortality, and the commingled CE-treated contact chicks (provided CE culture on day of hatch) average 35% S. gallinarum organ positive and 9% mortality during the same time period. These results are of importance to the poultry industry in geographic areas where poultry production is adversely affected by fowl typhoid and indicate that treating newly hatched chicks with this commercial CE culture may be a novel way of decreasing economic losses associated with this highly pathogenic organism.

Prophylactic administration of immune lymphokine derived from T cells of Salmonella enteritidis-immune pigs. Protection against Salmonella choleraesuis organ invasion and cecal colonization in weaned pigs.

Experiments involving 132 weaned piglets were conducted to evaluate the efficacy of a Salmonella enteritidis-immune lymphokine (PILK) derived from the T cells of Salmonella enteritidis (SE)-immunized pigs to protect weaned piglets from Salmonella choleraesuis (SC) infection. Fourteen-to-seventeen day-old piglets were weaned and randomly placed into 1 of 5 groups: (1) noninfected controls, (2) PILK 3X noninfected, (3) SC infected controls, (4) PILK 1X SC infected, and (5) PILK 3X SC infected. PILK was given orally either one time (PILK 1X) or three times (PILK 3X) over 14 days. One hour after the first PILK administration on day 0, piglets were orally challenged with 10(7) cfu of SC. Weights were recorded on day 0, day 7, and day 14. On day 14, pigs in groups 3, 4, and 5 were sacrificed and organs and lymph tissue were cultured for the presence of SC. Three replicates of this experiment were pooled and anlayzed. A significant reduction in the number of pigs positive for SC in the liver, lung, and spleen was found in group 5 (PILK 3X) when compared to group 3 (inf. cont. p < 0.001[). The number of SC positive cecal contents was dramatically reduced in group 5 group when compared to group 3, with the PILK 3X group showing 13% positive pigs versus 55.2% in the infected controls (p < 0.05). Weight gain over the 14 day study in the infected PILK 3X group (group 5) was found to be comparable to the gain observed in the group 1 (noninfected controls). The pigs receiving PILK 3X (group 2) with no SC challenge gained significantly more weight than all other groups, including the noninfected controls (group 1 p < 0.05[). The results of these experiments indicate that PILK protects against SC infection in weaned pigs while enhancing performance in the presence of an SC infection.

Effect of storage time and temperature on stallion sperm DNA and fertility.

We used the sperm chromatin structure assay (SCSA) to study the change in stallion sperm DNA susceptibility to denaturation after exposure of extended semen to three different storage temperatures (5, 20, or 37 degrees C) at 7, 20, 31, and 46 h. In addition, we compared the rates of sperm DNA denaturation in fertile and subfertile stallions. Among fertile stallions, spermatozoa stored at 20 and 37 degrees C showed a significant (P < 0.05) rise in the SCSA measures (Mean(alpha1), S.D.(alpha(t)), and percent cells outside the main population-COMP(alpha(t))) overtime, with the degree of rise being more dramatic at 37 degrees C. Over all stallions, samples stored at 5 degrees C showed no significant (P > 0.05) changes in the SCSA values measured over time, indicating maintenance of chromatin quality for up to 46 h. The COMP(alpha(t)) from stallions classified as subfertile showed an increased susceptibility to denaturation or decline in chromatin quality between 20 and 31 h when stored at 5 degrees C; however, spermatozoa from fertile stallions did not change during the time intervals analyzed. These data suggest that sperm DNA from some subfertile stallions may decline at a greater rate than spermatozoa from fertile stallions when exposed to similar storage conditions.

Relationship between stallion sperm motility and viability as detected by two fluorescence staining techniques using flow cytometry.

Relationships between sperm motility parameters and viability were evaluated using two fluorescent staining techniques in fresh extended semen (fresh and after 24 h storage at 5 degrees C) that had various concentrations of dead sperm added to simulate different levels of viable and nonviable sperm. Both protocols incorporated SYBR-14 and propidium iodide (PI) while the second protocol added the mitochondrial probe JC-1. The relationship between total sperm motility and percent viable sperm was high between staining protocols (r = 0.98). Time (0 h versus 24 h, P<0.0001) and treatment (0, 10, 25, 50, and 75% nonviable sperm, P<0.0001) affected percent total sperm motility and percent viable sperm for both staining protocols. Actual percent viable sperm for each time and treatment did not differ from expected values.

Functional responses of neonatal chicken and turkey heterophils following stimulation by inflammatory agonists.

OBJECTIVE: To determine functional responses of neonatal chicken and turkey heterophils to various inflammatory agonists. ANIMALS: 100 one-day-old chickens and turkeys. PROCEDURE: Blood heterophils were isolated and stimulated for 30 minutes at 39 C with ionomycin, phorbol myristate acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucyl-phenylalanine (FMLP). Functional responses (shape change, adherence, phagocytosis, influx of intracellular calcium, and oxidative burst) of stimulated heterophils were measured and compared with responses of unstimulated (control) heterophils. RESULTS: Turkey and chicken heterophils did not respond to FMLP stimulation. Stimulation of chicken and turkey heterophils with ionomycin resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, intracellular calcium concentration, and oxidative burst. Turkey heterophils did not respond to PMA stimulation, whereas stimulation of chicken heterophils with PMA resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, and oxidative burst but not intracellular calcium concentration. Stimulation of chicken and turkey heterophils with OZ resulted in significant increases in oxidative burst. CONCLUSIONS: Mechanisms regulating initiation of heterophil activation in neonatal chicken and turkey heterophils are consistent with those described for heterophils isolated from mature birds. The biochemical and cytoskeletal systems of neonatal avian heterophils undergo functional alterations following stimulation with inflammatory agonists. CLINICAL RELEVANCE: Understanding heterophil activation and regulation should eventually lead to methods for controlling bacterial diseases in poultry.

Lymphokine-augmented activation of avian heterophils.

Heterophils are important mediators of innate resistance in poultry, especially in young birds that have not yet developed an acquired immune response. Invasion of the intestinal mucosa by Salmonella spp. initiates the recruitment of large numbers of heterophils to the lamina propria. Thus, the heterophilic response can control, but not eliminate, bacterial numbers in the bird until development of acquired immunity. Unfortunately, chicks and turkey poults are highly susceptible to Salmonella infections during the first 4 d posthatch due to the functional immaturity of both the innate and acquired immune systems. We have previously shown that the administration of Salmonella enteritidis (SE)-immune lymphokines (ILK) into either 18-d-old developing embryos or day-of-hatch chicks and poults conferred increased resistance to SE organ invasion. In this review, we present evidence that the protection induced by ILK is mediated by vigorous recruitment and activation of heterophils. These activated heterophils migrate rapidly to the site of bacterial invasion where they phagocytize and kill the SE. Specifically, in vitro studies demonstrate an enhancement of functional activities of the heterophils including chemotaxis, adherence, phagocytosis, and bacterial killing. In addition, during the activation process, membrane expression of adhesion molecules rapidly changes from L-selectins to beta2 integrins (CB11b/CD18) on the cells that become activated. These results further demonstrate the validity of preventive activation in poultry to induce the migration of large numbers of activated phagocytic cells to the site of infection by a pathogenic organism. Importantly, this immunopotentiation of the inflammatory response by ILK, as described here, induces the functional maturation of heterophils during the first 4 d posthatch.

Administration of Salmonella enteritidis-immune lymphokine to day-old turkeys by subcutaneous, oral, and nasal routes: A comparison of effects on Salmonella enteritidis liver invasion, peripheral blood heterophilia and heterophil activation.

Previous experiments have shown that the administration by intraperitoncal (i.p.) injection of Salmonella enteritidis-immune Iymphokines (LK) derived from chicken splenic T cells protect both chicks and turkey poults from Salmonella enteritidis (SE) organ invasion. This protection was mediated by the avian heterophil. The present experiments evaluated the ability of SE-immune Iymphokines derived from a virally transformed chicken T cell line (VILK) in protecting day-old turkeys against SE liver invasion, inducing peripheral blood heterophilia, and functionally activating heterophils when delivered by subcutaneous (s.c), oral (p.o.) or intranasal (i.n.) routes when compared to i.p. injection. All routes of administration of VILK showed dramatic reductions in SE liver invasion (P < 0.01) and significant elevations in the number of heterophils in the peripheral blood (P < 0.01). The rise in peripheral blood heterophils was accompanied by a significant increase in the functional activity of the heterophils. Chemotactic movement of heterophils from all VILK groups was two to three times that observed in the control heterophils using chicken serum and recombinant human interleukin-8 (rhuIL-8) as the chemoattractants (P<0.01). The number of heterophils phagocytizing SE and the number of bacteria per heterophil were significantly higher in all VILK administered groups (P < 0.01). Adherence to bovine serum albumin (BSA) revealed significant increases in adherence of heterophils from all VILK administered groups when compared to control heterophils from poults (P<0.01). The results of these experiments clearly show that VILK delivered s.c, p.o. and i.n. is as effective in protecting day-old poults from SE invasion as an i.p. injection and that this protection is also mediated by the activation of an increased number of heterophils in the circulation.

Short-chain fatty acids affect cell-association and invasion of HEp-2 cells by Salmonella typhimurium.

This study demonstrates that the growth of S. typhimurium in Luria Bertani broth supplemented with acetate, propionate, butyrate, or a mixture of the three SCFA, affected cell-association and the ability to invade cultured HEp-2 cells. Cell-association and invasion was determined after growth for 4 h of growth in the presence of the SCFA at pH 6 and 7. The results suggest that the growth rate of the culture may have affected cell-association and invasion since accompanying the significant decrease in growth rate in the presence of SCFA at pH 6 was a decrease in cell-association and invasion. However, the results also suggest that the individual SCFA may play a role in modulating cell-association and the invasion phenotype and the regulation of cell-association and invasion by the SCFA was dependent on the concentration and the pH of the medium. Although the growth rates were similar for S. typhimurium in the SCFA mixture, butyrate (100 mM) and propionate (50 mM) at pH 6, differences in cell-association and invasion were observed among these cultures. Also, at pH 7, differences were observed among the SCFA treatments even though the growth rates were similar.