Rita B. Moyes

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

Hematopoietie 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 anti-human 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.

Differential Staining of Bacteria: Gram Stain

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram‐positive cocci, Gram‐negative cocci, Gram‐positive rods, and Gram‐negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. Curr. Protoc. Microbiol. 15:A.3C.1‐A.3C.8.

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.

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.

Fluorescent Staining of Bacteria: Viability and Antibody Labeling

This appendix presents several methods for using fluorescence to evaluate bacterial viability and to explore the cell surface for the presence of various antigens for diagnostic and taxonomic purposes. The use of fluorescent labeling will allow fast and accurate analysis and monitoring of microbial populations in ecological and clinical environments. Curr. Protoc. Microbiol. 15:A.3K.1‐A.3K.13.

Differential Staining of Bacteria: Capsule Stain

Bacterial capsules are composed of high‐molecular‐weight polysaccharides and/or polypeptides, and are associated with virulence and biofilm formation. Unfortunately, capsules do not stain well with crystal violet, methylene blue, or other simple stains. This unit describes two methods of capsule staining. The first is a wet‐mount method using india ink; the capsule is visualized as a refractile zone surrounding a cell. The second is a direct‐staining dry‐mount method that precipitates copper sulfate and leaves the capsule as a pale blue zone. Both methods are easily performed within ∼5 min. Curr. Protoc. Microbiol. 15:A.3I.1‐A.3I.4.

Differential Staining of Bacteria: Endospore Stain

Endospore production is a very important characteristic of some bacteria, allowing them to resist adverse environmental conditions such as desiccation, chemical exposure, extreme heat, radiation, etc. The identification of endospores is also very important for the clinical microbiologist who is analyzing a patients body fluid or tissue—there are not that many spore‐forming genera. In fact, there are two major pathogenic spore‐forming genera, Bacillus and Clostridium, together causing a number of lethal diseases—botulism, gangrene, tetanus, and anthrax, to name a few. Curr. Protoc. Microbiol. 15:A.3J.1‐A.3J.5.

Differential Staining of Bacteria: Acid Fast Stain

Acid‐fastness is an uncommon characteristic shared by the genera Mycobacterium (Section 10A) and Nocardia. Because of this feature, this stain is extremely helpful in identification of these bacteria. Although Gram positive, acid‐fast bacteria do not take the crystal violet into the wall well, appearing very light purple rather than the deep purple of normal Gram‐positive bacteria. Curr. Protoc. Microbiol. 15:A.3H.1‐A.3H.5.

Preliminary Staining of Bacteria: Negative Stain

Negative staining is one of the many staining techniques that can be employed for viewing of bacterial cell morphology and size. The advantages of the negative stain include the use of only one stain and the absence of heat fixation of the sample. Negative staining employs the use of an acidic stain and, due to repulsion between the negative charges of the stain and the bacterial surface, the dye will not penetrate the cell. In negative staining, the results yield a clear cell with a dark background. Curr. Protoc. Microbiol. 15:A.3F.1‐A.3F.5.

Differential Staining of Bacteria: Flagella Stain

Bacterial flagella are appendages used for motility. Their presence is a useful tool for identification and differentiation of prokaryotes. Since flagella are too thin to be seen by compound light microscopy, staining methods employ the use of a mordant (often tannic acid) to make them thick enough to see using an oil immersion objective. Two protocols are described. Basic Protocol 1 is a modified Leifson method and is the one that many microbiologists have adapted. Basic Protocol 2 is a wet‐mount stain using a Ryu stain and is included because the stain is stable at room temperature. Both of these methods are fairly time‐consuming, taking from 15 to as long as 60 min to perform. Curr. Protoc. Microbiol. 15:A.3G.1‐A.3G.5.