Use of lipopolysaccharide (LPS) as a positive control for the evaluation of immunopotentiating drug candidates in experimental avian colibacillosis models.

The aetiologic agent of avian colibacillosis is Escherichia coli. Colibacillosis is a disease that causes mortality and production performance problems in chickens which results in economic losses for the poultry industry. It will be increasingly important for scientists to identify novel solutions that can be implemented which will provide poultry producers with a tool to manage this economically important disease. The purpose of this investigation was to determine whether lipopolysaccharide (LPS) could be used as a positive control to evaluate novel chemistries for immunopotentiator activity in battery or floor-pen avian colibacillosis models in chickens. In the battery study, subcutaneous administration of LPS to one-day-old broiler cockerels caused a significant reduction (P < 0.003) in all parameters of colibacillosis evaluated, i.e. mean air sac lesion scores, per cent air sac lesions, E. coli re-isolation and per cent mortality. However, in the floor-pen study, subcutaneous administration to one-day-old broiler chicks resulted in a numerical, but not statistically significant reduction (P < 0.1) in mortality associated with colibacillosis. These data indicate that LPS can be used as a positive control to evaluate the efficacy of immunopotentiator drug candidates in avian colibacillosis models.

Effect of tylosin tartrate (Tylan Soluble) on cellular immune responses in chickens.

Although many antimicrobial agents have been reported to cause immunosuppression in animals, macrolide antibiotics enhance immune function. Tylosin is a macrolide antibiotic approved for the control of mycoplasmosis in poultry. The purpose of this investigation was to determine the effect of tylosin on cellular immune functions in chickens. There was no significant difference in adherent splenocyte chemotaxis between tylosin-treated and untreated (control) chickens. Tylosin increased splenocyte proliferation and splenocyte conditioned medium (CM) proliferative activity above control levels. Removal of adherent splenocytes before preparation of CM caused a reduction in CM proliferative activity. Tylosin also increased antitumor activity of splenocytes. These data are the first to suggest that the macrolide antibiotic, tylosin tartrate, has a modulatory effect in chickens on the immune parameters studied.

Effect of tylosin tartrate on humoral immune responses in chickens.

While many antimicrobial agents have been reported to cause immunosuppression in animals, macrolide antibiotics enhance the immune function. Tylosin tartrate is a macrolide antibiotic approved for the control of mycoplasmosis in poultry. The purpose of this investigation was to determine the effect of tylosin on the humoral immune functions in chickens. Three days after oral tylosin tartrate administration, 4- or 8-week-old chickens were immunized intravenously with carbolic acid-killed Brucella abortus bacterin or sheep red blood cells. Seven days (plasma antibodies titre) or 4 days (antibody forming cells) post-immunization, there was a significant increase in antibody production as well as in the numbers of antibody-producing cells in tylosin tartrate-administered chickens compared with the untreated controls. However, 3 days after tylosin tartrate administration, there was no difference in the distribution of T-lymphocyte subpopulations (CD4 or CD8 positive cells) or B lymphocytes (surface immunoglobulin positive cells) in either the peripheral blood or spleens or untreated control chickens.

Evaluation of Salmonella enteritidis-immune lymphokines on host resistance to Salmonella enterica ser. gallinarum infection in broiler chicks.

The prophylactic treatment of neonatal broiler chicks with lymphokines derived from S. enteritidis-immurazed chickens (SE-ILK) was evaluated for its effect on the birds' resistance to an experimental infection S. enterica ser. gallinarum (SG). On the day of hatch, chicks were injected intraperitoneally with either SE-ILK, control non-immune lymphokines (NILK), or were left untreated. Thirty minutes later, all chicks were orally gavaged with either 10(4) colony forming units (CFU) or 10(6) CFU SG. The chicks were observed twice daily for 10 days for morbidity and mortality. Chicks that died during the experiment had their livers cultured for SG. The survivors were killed and their livers, spleens and caecal tonsils cultured for SG. The prophylactic treatment of chickens with SE-ILK induced significant protection against extraintestinal SG infection when compared to the NILK-treated or non-treated controls as evidenced by: (1) a significant reduction (P< 0.005) in the mortality of chicks challenged with either 10(4) and 10(6) CFU SG; (2) an increased average weight gains of chicks challenged with either 10(4) and 10(6) CFU SG; and (3) a significant (P< 0.001) reduction in the number of chicks with organs culture-positive for SG. The results suggest that the prophylactic administration of SE-ILK can confer non-specific protection to chicks against a pathogenic species of Salmonella resulting in reduced morbidity, mortality, and organ infectivity caused by SG infections of broiler chicks, while enhancing performance during the first 10 days of Ufe.

Characterisation of colony-stimulating activity in the avian T cell-derived factor, Salmonella enteritidis-immune lymphokine.

This investigation was designed to characterise the specific cytokine activity from the conditioned medium of concanavalin A-stimulated avian T cells derived from Salmonella enteritidis-immune chickens, S enteritidis-immune lymphokine (ILK). Studies were designed to determine first, whether colony-stimulating activity was present in ILK, second, the type(s) of colonies from the bone marrow that were supported in vitro by the potential colony-stimulating factors in ILK and, third, whether colony-stimulating activity was present in serum from chicks treated with ILK and challenged with S enteritidis, and to use physicochemical treatment as a means of identifying the potential colony-stimulating factor(s) in ILK. Both ILK alone and serum from chicks treated with ILK and challenged with S enteritidis caused significant increases in the number of colony-forming units (CFU) from the bone marrow in vitro. After 10 days of incubation, ILK alone supported the in vitro growth of granulocytic bone marrow colonies. The colony-stimulating activity from serum derived from chicks treated with ILK and challenged with S enteritidis peaked two hours after the challenge. When ILK was either heated at 100 degrees C or treated with trypsin or acid and then injected into chicks, all the chicks responded with significant increases in circulating polymorphonuclear leucocytes (PMNs). However, when assayed for in vitro colony-stimulating activity, only trypsinisation destroyed the activity in ILK. The results indicate that a colony-stimulating factor which preferentially supported the growth of granulocytic bone marrow colonies was present in ILK and that the factor was stable to heat and acid but sensitive to trypsin.

Efficacy of Salmonella enteritidis (SE)-immune lymphokines from chickens and turkeys on SE liver invasion in one-day-old chicks and turkey poults.

We have shown previously that increased resistance to Salmonella enteritidis (SE) organ infectivity in 1-day-old chicks was conferred by the immunoprophylactic administration of SE-immune lymphokines (SEILK). These lymphokines have been found to be present in the cell culture media of concanavalin A-stimulated splenic lymphocytes obtained from SE-immunized chickens. In the present study we evaluated whether turkeys also produced SEILK and whether these lymphokines could protect 1-day-old chicks and turkey poults against SE liver invasion. In addition, we tested the ability of our original chicken SEILK to reduce SE liver invasion in turkey poults. Day-of-hatch chicks and turkey poults were injected intraperitoneally with immune lymphokines of either chicken or turkey origin. One hour later the birds were challenged per os with SE, and 20 hours later their livers were examined by bacteriological methods for the presence of SE. We found that SEILK induced from the splenic lymphocytes of SE-immunized turkeys reduced SE liver invasion in both chicks and turkey poults. Conversely, we also determined that SEILK produced by chicken splenic lymphocytes conferred protection against invasion by SE in turkey poults. This research is the first report of the production of SEILK in turkeys and also the first report on the cross-species activity of these effector molecules in chickens and turkeys.

Interaction of dexamethasone and Salmonella enteritidis immune lymphokines on Salmonella enteritidis organ invasion and in vitro polymorphonuclear leukocyte function.

We used an anti-inflammatory dose of dexamethasone (DEX) and Salmonella enteritidis (SE)-immune lymphokines (ILK) followed by oral SE challenge to chicks to determine the effects of these treatments on SE organ invasion and in vitro function of PMNs derived from peripheral blood. Endpoints included percent protection against SE organ invasion, numbers of peripheral blood PMNs, and in vitro PMN adherence, chemotaxis, and SE killing. SE organ invasion was significantly reduced in chicks treated with either ILK alone or DEX + ILK compared to controls. Chicks treated with either DEX alone or DEX + ILK responded with a significant increase in numbers of peripheral blood PMNs as compared to controls, while numbers of PMNs in the peripheral blood from chicks treated with ILK alone were not significantly increased. PMN adherence and percent SE killing by PMNs derived from chicks treated with either ILK alone or DEX + ILK were significantly increased compared to controls. Chemotaxis of PMNs derived from chicks treated with either ILK alone or DEX alone significantly increased 2-fold over control levels. Interestingly, chemotaxis of PMNs derived from chicks that received DEX + ILK was similar to controls. Generally, ILK abated the anti-inflammatory effects of DEX on PMNs in these assays, except for chemotaxis. We interpret these data to suggest that ILK may confer protection to chicks against the early phase of SE organ invasion by inducing an inflammatory response predominated by activated PMNs.

In vivo activation of heterophil function in chickens following injection with Salmonella enteritidis-immune lymphokines.

We have previously shown that increased resistance to Salmonella enteritidis organ infectivity in day-old chicks was conferred by the immunoprophylactic administration of S. enteritidis-immune lymphokines (ILK). This resistance was associated with a significant increase in the number of circulating heterophils 4 h after ILK injection. The objective of the present study was to evaluate heterophil function following the administration of ILK in day-old chicks. Significant increases (P < 0.001) in adherence, chemotaxis, and phagocytosis of S. enteritidis were found with heterophils isolated from ILK-injected chickens compared to the heterophils isolated from birds injected with either pyrogen-free saline or lymphokines from non-immune T cells. After phagocytosis, the heterophils from the ILK-injected chickens were also able to kill significantly greater numbers of S. enteritidis more rapidly than did the heterophils from the saline-injected control birds (within 30 min, control cells killed 21.89% of the bacteria whereas ILK-treated cells killed 88.22%). We also found that the heterophils from the ILK-injected birds were more efficient killers of S. typhimurium, S. gallinarum, and E. coli. These results strongly suggest that the protection against S. enteritidis organ invasion induced by the prophylactic treatment of day-old chicks with ILK involves activated heterophils which migrate rapidly to the inflammatory stimulus where they phagocytize and kill the bacteria.

In ovo administration of Salmonella enteritidis-immune lymphokines confers protection to neonatal chicks against Salmonella enteritidis organ infectivity.

We previously reported that the prophylactic, intraperitoneal administration of supernatants from concanavalin A-stimulated T cells derived from Salmonella enteritidis (SE)-immune White Leghorn hens (i.e., SE-immune lymphokines or ILK), conferred protection to neonatal White Leghorn chicks against SE organ invasion. In the present study, we evaluated the effects of in ovo administration of ILK on hatchability, hatch weight, in vitro bactericidal activity of heterophils, and protection against SE organ invasion in neonatal White Leghorn chicks. On Day 18 of embryogenesis, injections were made into the amnion with either ILK or nonimmune ILK (NILK) or were not injected (untreated). On the day of hatch, whole blood was collected from 20 of the chicks per treatment group for heterophil isolation. All remaining chicks were orally challenged with 5 x 10(4) cfu SE. Twenty-four hours after SE challenge, organs (liver and spleen) from the chicks were cultured for SE. Hatchability of ILK- and NILK-treated chicks was not different from that of untreated chicks. Hatch weights of ILK-treated chicks were approximately 1 g less (P < .05) than that of NILK-treated or untreated chicks. In vitro bactericidal activity of peripheral blood heterophils derived from ILK-treated chicks was increased (P < .05) above activity from heterophils derived from NILK-treated or untreated chicks. Organ invasion with SE was markedly and significantly decreased in the ILK-treated chicks as compared with chicks treated with NILK or untreated chicks. These results suggest that in ovo administration of ILK confers protection to neonatal chicks against SE organ infectivity at hatch.

Characterization of the pattern of inflammatory cell influx in chicks following the intraperitoneal administration of live Salmonella enteritidis and Salmonella enteritidis-immune lymphokines.

We characterized the inflammatory cell influx in day-old chicks induced by the i.p. administration of live Salmonella enteritidis (SE) and lymphokines from concanavalin A-stimulated SE-immune T lymphocytes (ILK). An i.p. injection of ILK along with 5 x 10(3) cfu SE increased the survival rate of chicks 48 h later from 70% (ILK-treated controls) compared with 25% (saline-treated). The injection of both the ILK and live SE (but not formalin-killed SE) resulted in an increased influx of inflammatory heterophils into the peritoneum that peaked at 4 h after the injections with no increase in peritoneal macrophages. The heterophil accumulation was not influenced by polymyxin B, but was sensitive to heat treatment (100 C for 1 h) of the ILK, suggesting that lipopolysaccharide (LPS) did not contribute to the induced accumulation of heterophils. Treatment of the chicks with nordihydroguaiaretic acid or indomethacin did not abrogate the induced heterophil accumulation, suggesting that arachidonic acid metabolites were not involved in the SE/ILK-induced accumulation of peritoneal heterophils. The results from the current studies indicate that 1) ILK-mediated resistance to SE-induced mortality is mediated by a rapid influx of inflammatory heterophils to the site of infection; 2) live SE, during invasion, are vital for the site-directed migration of the heterophils; and 3) the mechanisms of induced heterophil accumulation are unknown but involve neither LPS nor arachidonic acid metabolites.

Comparison of prophylactic and therapeutic efficacy of Salmonella enteritidis-immune lymphokines against Salmonella enteritidis organ invasion in neonatal Leghorn chicks.

Investigations in our laboratories have indicated that when Salmonella enteritidis (SE)-immune lymphokines--supernatants from concanavalin-A-stimulated T cells derived from SE-immune adult chickens--were administered intraperitoneally to 1-day-old chicks before SE challenge, they conferred protection against SE organ invasion within 24 hr. This resistance mediated by SE-immune lymphokines was associated with a concomitant increase in peripheral blood polymorphonuclear leukocytes that peaked 4 hr after SE challenge. In the present study, we evaluated efficacy of SE-immune lymphokines in protecting chicks against SE organ invasion and alterations in peripheral blood polymorphonuclear leukocyte counts. Administration of SE-immune lymphokines to chicks either 30 min or 6 days before SE challenge caused a significant reduction in SE organ invasion. However, when SE-immune lymphokines were administered 2 days after SE challenge, there was no reduction in SE organ invasion. Both prophylactic (before SE challenge) and therapeutic (after SE challenge) administration of SE-immune lymphokines caused a significant increase in numbers of peripheral blood polymorphonuclear leukocytes. Results from these studies suggest that SE-immune lymphokines have potential value as an effective prophylactic but not as a therapeutic modulator of early resistance to SE organ invasion in neonatal leghorn chicks.

Dynamics of avian inflammatory response to Salmonella-immune lymphokines. Changes in avian blood leukocyte populations.

Investigations in our laboratories have indicated that an increased resistance to SE organ infectivity in chicks was conferred by the immunoprophylactic administration of SE-immune lymphokines (SE-ILK). This resistance was associated with an increase in the lamina propria thickness due to a marked infiltration of inflammatory polymorphonuclear cells (PMNs). In the present study, we determined whether the hematological profile of SE-ILK-treated chicks might reflect changes that are associated with the protection against organ invasion by SE. As protection has been observed in previous studies within 24 h of SE-ILK administration, we evaluated alterations in the circulating leukocyte profile in 1-day-old Leghorn chicks during this time period. We also determined whether the alterations in the peripheral blood leukocytes correlated with the increased protection against SE organ invasion induced by the SE-ILK. Within 4 h after an intraperitoneal injection of SE-ILK and challenge with SE, the number of circulating leukocytes increased significantly (P < 0.05) from all of the other treatment groups. The number of circulating PMNs was found to account for more than 80% of the increase in the number of circulating leukocytes. Using correlation analysis, we found a strong association between the number of circulating PMNs and the protection induced by SE-ILK against SE organ invasion. These studies associate the expansion of the available pool of circulating PMNs and the expression of innate resistance to organ invasion by SE.

Comparison of the polymerase chain reaction using genus-specific oligonucleotide primers and microbiologic culture for the detection of Salmonella in drag-swabs from poultry houses.

Drag-swab samples were collected from 18 poultry houses at 9 broiler farms. Fifty drag-swab samples were tested for Salmonella by microbiologic culture using selective enrichment and the polymerase chain reaction (PCR) with oligonucleotide primers specific for all members of the genus Salmonella. Drag-swab samples were tested for Salmonella using PCR before and after enrichment. Only one sample was positive by PCR prior to enrichment. Forty-seven of the drag-swabs samples tested after enrichment were positive for Salmonella using PCR, and 29 were positive by microbiologic culture. All but one of the culture-positive samples were positive by PCR; this discordant sample was classified as indeterminate by PCR. Salmonella was identified in houses from all nine farms by PCR and eight of nine farms by microbiologic culture. Salmonella was found in all 18 houses by PCR and in 15 of 18 houses by microbiologic culture. In this study, PCR was significantly (P < .001) more sensitive than culture for environmental monitoring of Salmonella using drag-swabs.

Genus-specific detection of salmonellae in equine feces by use of the polymerase chain reaction.

Members of the genus Salmonella were identified in feces from horses, using the polymerase chain reaction (PCR) and genus-specific oligonucleotide primers. Feces from healthy horses were determined to be culture-negative for Salmonella spp. Fecal samples were inoculated with known numbers of colony-forming units (CFU) of S anatum, S derby, S enteritidis, S heidelberg, S newport, and S typhimurium. The DNA was extracted from fecal samples and amplified by PCR, using genus-specific primers. Sensitivity of the assay extended to 10(3) CFU of Salmonella sp/g of feces; sensitivity of microbiologic culture with enrichment extended to 10(2) CFU of Salmonella sp/g of feces. Feces that were not inoculated with Salmonella spp were negative by the PCR. Detection of Salmonellae in feces was possible, using the PCR, within 10 to 12 hours from the time of submission of samples.

Heterophils are decisive components in the early responses of chickens to Salmonella enteritidis infections.

Chickens injected with 5-fluorouracil (5-FU) were found to have a three- to nine-fold reduction in circulating heterophils without a reduction in the number of circulating mononuclear cells. A 50% organ invasive dose (ID50) for orally and intravenously administered Salmonella enteritidis (SE) was established in the heteropenic chickens. When challenged orally, about 150-fold fewer SE cells were required for organ invasion in the 5-FU-treated chickens; whereas an intravenous challenge of the heteropenic chickens required about 4000-fold fewer bacteria for organ invasion than found in the control birds. Significant (P < 0.0001) SE-dose-dependent reductions in body weight with increased mortality and lesions in the eye, heart, and thymus were found in the 5-FU-treated birds versus the control birds. Alternatively, the control birds had significantly more intestinal lesions than the 5-FU-treated birds. No consistent significant hepatic lesions were observed in either treatment group. Under the conditions used in these experiments, 5-FU treatment of chickens caused otherwise subclinical SE infections to rapidly become clinical infections with more severe extraintestinal organ infections, whereas the control chickens had infections that were primarily restricted to the intestine. These findings suggest that the heterophil is extremely important in controlling both initial SE organ invasion and subsequent disease pathogenesis in chickens.

Detection of Salmonella enteritidis in feces from poultry using booster polymerase chain reaction and oligonucleotide primers specific for all members of the genus Salmonella.

Salmonella enteritidis was identified in feces from hens using the polymerase chain reaction (PCR) and oligonucleotide primers specific for all members of the genus Salmonella. Feces from specific-pathogen-free Leghorn hens were determined to be negative for Salmonella by microbiological culture and by the PCR. Fecal samples were inoculated with known numbers of colony-forming units of S. enteritidis. The DNA was extracted from fecal samples and amplified by the PCR using genus-specific primers. Salmonella were detected in all samples known to be positive; the sensitivity of the assay extended to 1 cfu of S. enteritidis/g feces. Feces that were not inoculated with Salmonella were negative. Microbiological culture was less sensitive than the PCR assay; results of culture of feces with less than 10(2) cfu/g were negative. Although S. enteritidis was used in this study, the oligonucleotide primers used in this study have been previously demonstrated to be genus-specific for Salmonella.

Salmonella enteritidis immune leukocyte-stimulated soluble factors: effects on increased resistance to Salmonella organ invasion in day-old Leghorn chicks.

Cytokines, derived from either concanavalin A-stimulated Salmonella enteritidis-immune chicken T lymphocytes [SE-immune Lymphocyte Stimulated Soluble Factor (LSSF)] or lipopolysaccharide-stimulated SE-immune chicken macrophages [SE-immune Macrophage Stimulated Soluble Factor (MSSF)], were evaluated for their ability to increase resistance to SE organ invasion in day-old Leghorn chicks. In Trial 1, day of hatch chicks were injected i.p. with either SE-immune LSSF or SE-nonimmune LSSF (control). In Trial 2, chicks were similarly injected with either SE-immune MSSF, SE-nonimmune MSSF, or SE-immune LSSF (positive control). Thirty minutes postinjection, all chicks were gavaged with an invasive dose of SE. Twenty-four hours later, livers and spleens from all chicks were cultured for SE. In Trial 1, SE-immune LSSF caused a rapid and marked protection (P < .01) against SE infection as determined by the number of chicks that were culture positive regardless of challenge dose. In Trial 2, SE-immune MSSF was not associated with protection against SE organ infection. These experiments demonstrate that SE-immune LSSF, but not MSSF, are able to confer protection against SE organ invasion in day-old Leghorn chicks. Thus, it appears that the stimulated immune T cell, and not the macrophage, is responsible for producing the soluble products that protected the chicks.

Genus-specific detection of salmonellae using the polymerase chain reaction (PCR).

Oligonucleotide primers for the polymerase chain reaction (PCR) that enable genus-specific detection of members of the genus Salmonella were developed. The primers amplify a 496-bp genetic sequence of members of the genus Salmonella. Amplification of DNA extracted from all other genera of the family Enterobacteriaceae and various other gram-positive aerobic and anaerobic bacteria yielded negative results. Applications of the PCR using these genus-specific primers are discussed.