Cellular effects of T-2 mycotoxin on two different cell lines.

The effects of T-2 toxin on protein synthesis, respiratory chain activity of the mitochondria, cell lysis and toxin-cell binding were compared in the toxin-sensitive bovine kidney cell line (MDBK) and in the toxin-resistant Chinese hamster ovary cell line (CHO). Protein synthesis and mitochondrial activity were 10-fold less sensitive in CHO cells as compared to MDBK (50% inhibition = 10-15 ng/ml vs. 1-1.5 ng/ml, respectively). Lytic activity, as determined by release of 51Cr from cells incubated at 4 degrees C, was not detected at 5 h or 24 h in either of the cell lines. However, at 24 h, CHO cells released less 51Cr than non-toxin-exposed controls, indicating that some membrane interaction does occur. Both cell lines equally bound [3H]T-2 toxin at 4 degrees C. At 37 degrees C, the MDBK cells take up twice as much [3H]T-2 toxin at 2 h and 6 h. These results indicate that T-2 toxin mediates a number of effects on the cell at the level of the membrane, protein synthesis and probably mitochondrial activity toward 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction.

Targeting of mouse erythrocytes by band 3 crosslinkers.

Chemical conditions of crosslinking mouse erythrocytes with BS3 and DTSSP have been studied. These two crosslinking reagents seem to react with band 3 protein in mouse erythrocytes membrane. Extent of crosslinking is dependent on the concentration of the reagent used. Similar cell volumes were observed in crosslinked erythrocytes with respect to control erythrocytes. In vivo behaviour of these modified erythrocytes revealed prominent targeting of crosslinked erythrocytes to liver. This effect is clearly evident when concentrations of 5 mM BS3 or DTSSP were used and can be dependent of reagent concentration. Consequently, from our results BS3 and DTSSP can be considered as very useful tools to control and modulate targeting of crosslinked erythrocytes.

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.

Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis.

Hapten-carrier protein conjugates were made using five different small haptens (MW < 1000), two carrier proteins and two methods of conjugation. Nondenaturing agarose gel electrophoresis was used to demonstrate that when as few as two molecules of a small hapten are attached to the carrier, the conjugate band migrates differently from that of the carrier alone or of the coupling reagent-treated carrier. Furthermore, the direction of the change in migration of each conjugate correlates with the change in charge which occurs upon attachment of the hapten to the carrier.

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.

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.

Cytotoxic effect of T-2 mycotoxin on cells in culture as determined by a rapid colorimetric bioassay.

We developed a colorimetric assay for determining metabolic activity (viability) of cells exposed to toxic agents. This system is based on the ability of mitochondrial enzymes in viable cells to modify a tetrazolium salt into a blue formazan product that can be detected spectrophotometrically at 570 nm. The assay works equally well for mammalian and insect cell lines and at 48 hr color formation is linear over a cell input range of 1.56-50 X 10(4) cells/ml. The inhibitory effects of T-2 mycotoxin on tetrazolium cleavage in L929 cells is comparable to that observed for protein and DNA synthesis (50% inhibition = 6-8 ng/ml). Using this system to analyze the lethal effect of T-2 toxin on cells from various animal species, it was found that bovine cells were the most sensitive (50% inhibition at 2.2 ng/ml) while hamster cells were the most resistant (50% inhibition at 26.2 ng/ml). Murine cells exhibited intermediate sensitivity (50% inhibition at 10.9 ng/ml). Variable toxin susceptibility was also observed among different cell types. Lymphocytes were 3-fold more sensitive to the T-2 inhibitory effects than comparable tissue culture cell lines. These data indicate that the colorimetric assay system could have broad applications in toxicological studies. Further, the observed differences in species sensitivity may provide insight into the primary mechanism of the T-2 toxin-cell interaction that ultimately leads to cell death.

Detection of the lethal effects of T-2 mycotoxin on cells using a rapid colorimetric viability assay.

A colorimetric method of determining cell viabilities in cultured cells is described. The system is based on the ability of mitochondrial enzymes in live but not dead cells to chemically reduce a tetrazolium salt (MTT) into a colored formazan dye which can be detected at 570 nm using a multiwell scanning spectrophotometer. 48 h Chinese hamster ovary (CHO) cell cultures are used in the assay and the amount of colored product formed is directly proportional to cell number over a range of 0.39-12.5 X 10(4) cells/ml. The cytotoxic effects of T-2 mycotoxin can also be detected colorimetrically using this method. The toxin dose which inhibits formazan formation (50% endpoint = 14-16 ng/ml) is very comparable to that which inhibits cell viability (17 ng/ml), or protein and DNA synthesis (10 ng/ml). This system also works well with mitogen-stimulated primary lymphocyte cultures but these cells exhibit a much more sensitive response to T-2 effects having a 50% inhibition endpoint of 2 ng/ml. The assay is rapid to perform and gives a high degree of precision and could serve as a valid alternative to viability assays currently in use.

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.

Artificial feeding of Ornithodoros concanensis (Acari: Argasidae) nymphs on bovine blood and morphological changes in erythrocytes undergoing hemolysis in the tick midgut.

The morphology of bovine erythrocytes undergoing hemolysis in the midgut of the argasid tick Ornithodoros concanensis Cooley & Kohls was examined after feeding nymphal ticks artificially on parafilm and latex membranes. Percentage of successful feeding was significantly higher on parafilm (63%) than on latex (43%) membrane. However, there was no difference in the amount of blood taken per tick. Scanning electron microscopy was used to follow the morphological changes of red blood cells in the tick gut during an 80-h postfeeding period. Erythrocytes converted from normal discocytes to spiculated cells (echinocyte stage III) within 20 h. During the development of spicules (echinocytosis), erythrocytes lost membrane surface area by the release of microvesicles. At 30 h after feeding, the first spherocytes appeared and after 80 h, only smooth spheres of different sizes were present. Reasons for the observed modifications in red blood cell morphology are discussed.

Production and characterization of a monoclonal antibody against bovine haptoglobin and its use in an ELISA.

Haptoglobin (Hp) is the major acute phase reactant found in cattle. As such, it is an excellent indicator of early disease processes and could be used as a marker for pre-clinical illness in cattle. The production of monoclonal antibodies (mAbs) directed against bovine Hp and bovine hemoglobin is described. The anti-haptoglobin mAbs (Hap1, Hap2, Hap3) and the anti-bovine hemoglobin (Hb) mAb (BoHem1) were characterized and tested for cross-reactivity by means of enzyme-linked immunosorbent assay (ELISA) and immunoblotting analyses. Additionally, the development of an ELISA based on an anti-haptoglobin mAb is discussed.

Differential expression of adhesion molecules by chicken heterophils activated in vivo with Salmonella enteritidis-immune lymphokines.

Chicken heterophils activated in vivo following the intraperitoneal (i.p.) administration of Salmonella enteritidis-immune T lymphokines (SE-ILK) have been implicated in the protection against SE organ invasion. SE-ILK induces a heterophilia and directly (or indirectly) activates the granulocytes. The invasion of SE provides the secondary signal for directing activated heterophils to the site of bacterial invasion. We examined the mechanism of adherence within the avian heterophil system using an in vitro bovine serum albumin (BSA) matrix in which neutrophil adherence is primarily CD11/CD18 integrin mediated in mammalian systems. Activated heterophils displayed a four-fold increase in receptor-mediated adherence in vitro to BSA-coated slides as compared to control heterophils from PBS-injected birds. The increased adherence of activated heterophils can be partially blocked by either anti-alpha M (CD11b) or anti-beta 2 (CD18) antibodies in a dose dependent manner. Anti-alpha 3 (CD49c) antibody partially blocked adherence of both normal and activated cells. Fluorescence-activated cell scanning (FACS) analysis of the heterophils shows that both control and SE-ILK-activated heterophils collected at 4 h post injection with SE-ILK or PBS display similar amounts of integrin alpha 3 on their surface. This integrin is constitutively expressed and is responsible for the in vitro adherence of both groups. However, antibodies to the Mac-1 complex (CD11b/CD18) block only the adherence of SE-ILK-stimulated heterophils. Thus, the CD11b/CD18 heterodimer is apparently up regulated in response to the injected SE-ILK and plays a major role in the adherence of activated heterophils. Our studies in chickens parallel human and mouse studies showing the importance of the beta 2 integrins in adherence of activated cells.

Neutralization of G-CSF inhibits ILK-induced heterophil influx: granulocyte-colony stimulating factor mediates the Salmonella enteritidis-immune lymphokine potentiation of the acute avian inflammatory response.

Hematopoietic colony stimulating factors (CSF) regulate the growth and development of phagocytic cell progenitors and also augment functional activation of phagocytes. Granulocyte-CSF (G-CSF) is the CSF that acts specifically upon granulocyte progenitor cells and mature granulocytes. We have shown that lymphokines (ILK) from T cells of birds immunized against Salmonella enteritidis (SE) induce a granulocytic (PMN) inflammatory response in chicks challenged with SE. This inflammatory response was characterized by: (a) a dramatic emigration of granulocytic cells from the bone marrow into the peripheral blood, (b) an enhancement of the biological functions of the circulating PMNs, and (c) a directed influx of these activated PMNs to the site of bacterial invasion. In the current study, we determined the presence of G-CSF in ILK by Western blot analysis using a goat polyclonal antihuman G-CSF antibody (Ab). Using this Ab, we then evaluated the role of G-CSF in the ILK-induced protective inflammatory response in chickens against SE. Pretreatment of ILK with the Ab totally abolished the colony-stimulating activity of the ILK. Furthermore, Ab treatment of ILK resulted in: (a) an elimination of the ILK-induced peripheral blood heterophilia with a dramatic inhibition of ILK-mediated protection against SE organ invasion and (b) an elimination of accumulation of inflammatory PMNs in the peritoneum with subsequent decrease in the survival rate of chicks challenged i.p. with SE. Taken together these studies demonstrate for the first time the contribution of G-CSF to avian PMN activation and the immunoprophylaxis of SE infection by ILK in neonatal chickens.

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.

Early Salmonella challenge time and reduction in chick cecal colonization following treatment with a characterized competitive exclusion culture.

Broiler chicks were treated by oral gavage on the day of hatch with a continuous-flow competitive exclusion culture (PREEMPT). At 4 h, 1 day, or 2 days posttreatment, chicks were challenged by oral gavage with 10(2) or 10(4) Salmonella CFU to determine the effects of challenge time on Salmonella cecal colonization. Cecal propionic acid concentrations in two trials increased (P < or = 0.001) within 1 day posttreatment in chicks given PREEMPT, and the increases were indicative of the establishment of the PREEMPT bacteria. Salmonella cecal populations decreased (P < or = 0.001) on average 6 log10 units in these two trials in chicks challenged 4 h posttreatment with 10(4) Salmonella CFU. In a third trial propionic acid did not increase significantly until 2 days after treatment, and there was no decrease in Salmonella colonization when chicks were challenged at 4 h after treatment. However, there were decreases in that same trial when chicks were challenged at 1 and 2 days after treatment. The early establishment of PREEMPT followed by challenges with 10(2) and 10(4) Salmonella CFU resulted in 3% and 3%, respectively, of the ceca testing Salmonella-culture-positive, compared to 28% and 95%, respectively, culture-positive ceca in untreated chicks. The results from this study indicated that in most instances young broiler chicks can be protected against cecal colonization when challenged with 10(2) and 10(4) Salmonella CFU as early as 4 h posttreatment on the day of hatch with the PREEMPT bacteria.

Provision of lactose to molting hens enhances resistance to Salmonella enteritidis colonization.

Older leghorn hens, more than 50 weeks of age, were divided into three groups designated 1, unmolted controls; 2, molted; or 3, molted treated with lactose. Forced molt was induced by 14 days of feed removal. Lactose was provided to the hens in group 3 as 2.5% (wt/vol) of the daily drinking water. Each hen in all groups was challenged orally with 10(5) Salmonella enteritidis (SE) cells on day 7 of feed removal. The study was repeated in three replicated trials. The concentrations of acetic, propionic, and total volatile fatty acids (VFA) in the cecal contents of the molted hens in groups 2 and 3 decreased significantly (P < 0.05) on days 6 and 14 of molt compared with the unmolted controls. Forced molt had no apparent effect on pH or on the oxidation-reduction potential of the ceca. Compared to the unmolted controls, SE cecal and spleen and liver colonization was significantly increased (P < 0.05) in the molted hens in group 2. Compared to the molted hens in group 2, SE cecal and spleen and liver colonization was significantly decreased (P < 0.05) in two of three trials in the hens in group 3 provided with lactose. The results suggested that the increased susceptibility of molting hens to SE colonization may be associated with decreased fermentation and production of VFA by cecal bacteria or by a depletion of the number of VFA-producing bacteria present in the ceca. The results further suggest that providing lactose in the drinking water during molting may significantly enhance resistance to SE colonization.

Dosage titration of a characterized competitive exclusion culture to inhibit Salmonella colonization in broiler chickens during growout.

Broiler chicks were spray treated on the day of hatch with titrated dosages (10(6), 10(7), or 10(8) anaerobic CFU) of a characterized competitive exclusion culture (CF3) and challenged orally on day 3 with 10(4) CFU of Salmonella typhimurium. On day 10, cecal contents from control and CF3-treated chicks were cultured for S. typhimurium to determine the minimal efficacious dosage of the CF3 culture. The experiment was repeated in three replicated trials. Resistance to Salmonella cecal colonization was dosage related and progressively enhanced at the 10(7)- and 10(8)-CFU dosages compared with the 10(6)-CFU dosage. The 10(7)-CFU dosage was selected as the minimal effective dosage and evaluated for efficacy during a 43-day broiler growout study. Six hundred broilers were spray treated on the day of hatch and compared with 600 controls. One-half of the control and CF3-treated birds were challenged orally on day 3 with 10(4) CFU of S. typhimurium and designated "seeders." The remaining unchallenged birds were designated "contacts." Compared with the controls, the recovery of Salmonella cells from the ceca of the CF3-treated broilers was significantly decreased (P < 0.01) in the challenged seeders on days 21 and 43 of growout. Salmonella contamination of floor pen litter was significantly reduced (P < 0.05) in pens of CF3-treated birds compared with controls. The transmission of Salmonella cells from seeder to contact birds in the same pens was decreased significantly (P < 0.01). The results indicated that treatment of broiler chicks on the day of hatch with the 10(7)-CFU dosage of CF3 culture effectively increased resistance to S. typhimurium challenge during growout to market age.

Assessment of the long-term shedding pattern of Salmonella serovar choleraesuis following experimental infection of neonatal piglets.

In the United States, swine salmonellosis is most often attributed to infections by Salmonella serovar choleraesuis. As a host-adapted pathogen rarely found in nonswine sources, S. choleraesuis is thought to be spread primarily via horizontal transmission, with carrier animals playing an important role. Little has been reported regarding infection of neonatal piglets, particularly regarding their potential to become carriers. Evidence reported herein demonstrates that piglets experimentally infected by S. choleraesuis at 2 days of age were capable of shedding the pathogen for up to 85 days postinfection, at which time the study was concluded. This study also presents findings supporting the use of GN-Hajna as a preenrichment medium for the isolation of S. choleraesuis.

In vitro feeding of Psoroptes ovis (Acari:Psoroptidae).

An in vitro feeding test is described for Psoroptes ovis. The criterion for feeding response was established on the basis of coloration of mites by a red dye or by red blood cells. Feeding response was about 85% for female mites held off host cattle for 1 day. Mites preferred water plasma or serum and low-salt diets over whole blood. Feeding response was detected within 5 min of exposure to the diet, and maximum response was observed within 30 min of contact. Mites ingested an average 0.29 microliter of diet/100 mites in a single feeding. At temperatures between 10 degrees and 42 degrees C, feeding response was not significantly different.

Ultrastructure of Hepatozoon canis in the dog.

The ultrastructure of several stages of Hepatozoon canis found in dogs with clinically diagnosed infections was determined using transmission electron microscopy. Stages were found in skeletal muscle tissue that corresponded to the 'onion skin' cyst stage, as described at the light microscopic level, and were composed of an electron-transparent material that appeared to radiate from a central core. Larger cysts, walled off by fibroblasts, contained a transformed host cell located centrally within them. The parasitic stage within these transformed cells contained numerous organelles including mitochondria and Golgi apparatus, and was singularly nucleated. In cardiac muscle, a meront was observed which produced merozoites by ectomerogony. Adjacent to this meront was a granuloma containing merozoites within mononuclear phagocytes which may either serve as a reservoir of parasites for reinfection of the host or differentiate into the circulating gamont stage of the parasite. Gamonts were found within parasitophorous vacuoles inside circulating neutrophils. They had a condensed cytoplasmic appearance and were extremely electron dense with respect to other observed parasite stages. In vitro cultivation of parasitized neutrophils resulted in the appearance of a stage of the parasite with altered ultrastructure compared with gamonts found in circulating neutrophils. This stage was judged to be a possible gamete stage of the parasite.