The insect immune protein scolexin is a novel serine proteinase homolog

Scolexin is a coagulation-provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin-like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism.     

Biochemical characterization,developmental expression,and induction of the immune protein scolexin from Manduca sexta

The immune protein, scolexin, a bacteria-induced, larva-specific protein from Manduca sexta, was shown to exist in the hemolymph in two isoelectric forms designated herein as scolexin-1 and scolexin-2 (native M_r ～ 72 kd). These two charge isomers appeared to share the same amino acid composition. Scolexin is composed of two subunits (peptide M_r ～ 36 kd) that possess the same N-terminus. Scolexin-2 was subjected to glycosyl composition analysis, revealing the presence of galactose, glucose, mannose, xylose, and sialic acid residues. Hybridization of epidermal RNA with oligonucleotides deduced from the scolexin N-terminal sequence showed a continuous decline in mRNA following day 0 of the 5th larval instar. By employing in vitro protein labelling, it was found that organ cultures of the epidermis from immune larvae showed a greater ability over that of naive epidermal cultures to synthesize scolexin; these data reflected the inducible response seen in the hemolymph, and confirm other data indicating that the epidermis is an important site of scolexin biosynthesis. © 1995 Wiley-Liss, Inc.     

Growth and virulence of Candida albicans after oral inoculation in the chick with a monoflora of either Escherichia coli or Streptococcus faecalis

Balish, Edward (Syracuse University, Syracuse, N.Y.), and A. W. Phillips. Growth and virulence of Candida albicans after oral inoculation in the chick with a monoflora of either Escherichia coli or Streptococcus faecalis. J. Bacteriol. 91:1744-1749. 1966.-Bacterial protection against intestinal infection by Candida albicans was investigated in chicks with a monoflora of either Escherichia coli or Streptococcus faecalis. These animals were obtained by orally inoculating germ-free chicks (3 days old) with pure cultures of bacteria. Each bacterial species was established in large numbers in the gut of separate groups of animals within 24 hr of inoculation; these numbers were similar in chicks examined 34 days later, at which time all animals were killed. The numbers of bacteria from contents of the crop, small intestine, and ceca were similar in chicks with the E. coli monoflora. Comparable results were obtained in chicks with the S. faecalis monoflora, except for decreased numbers in the duodenum and jejunum. Some of the monoflora chicks (7 days old) were transferred into separate isolators, orally inoculated with C. albicans, and observed for 34 days. All chicks grew well and appeared healthy. However, examinations at autopsy revealed severe crop infections in chicks with a diflora containing S. faecalis. Preferential growth of hyphae (C. albicans) occurred in the lesions and throughout the gut. The numbers of S. faecalis in the gut were comparable to those found in unchallenged animals. Agglutinins against C. albicans were not detected in our test or control chicks. Chicks with a diflora containing E. coli and C. albicans had a few microscopic crop lesions containing small numbers of hyphae. C. albicans was well established in the gut of these animals, largely as the yeast form. The numbers of E. coli in the gut were similar to those in control chicks. Thus, it was concluded that E. coli provided protection against crop infection by C. albicans. In crop contents from unchallenged animals, chicks with S. faecalis monoflora were about pH 5, whereas birds with E. coli monoflora were about pH 7. The challenge did not greatly change the former value, and the latter was slightly decreased. In the crop of unchallenged birds, negative E(h) values were found in chicks with S. faecalis and positive E(h) values in those with E. coli. Challenge did not greatly change these values. These data on pH and E(h) were related to conditions for morphogenesis of C. albicans and virulence. No major difference in the concentrations of serum proteins was seen in chicks with E. coli or S. faecalis after challenge with C. albicans. Possible mechanisms of the protective effect of E. coli are discussed.     

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism.

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organism's pathogenesis.     

Changes in the Plasma Membrane Distribution of Rice Phospholipase D during Resistant Interactions with Xanthomonas oryzae pv oryzae

Phospholipase D (PLD; EC 3.1.4.4), which hydrolyzes phospholipids to generate phosphatidic acid, was examined in rice leaves undergoing susceptible or resistant interactions with Xanthomonas oryzae pv oryzae. RNA analysis of leaves undergoing resistant interactions revealed different expression patterns for PLD over 5 days relative to control plants or those undergoing susceptible interactions. By using an activity assay and immunoblot analysis, we identified three forms of PLD (1, 2, and 3). PLD 1 was observed only at 1 day after tissue infiltration. PLDs 2 and 3 were detected up to 3 days in all interactions. Immunoelectron microscopy studies revealed PLD to be associated predominantly with the plasma membrane. In cells undergoing a susceptible response, PLD was uniformly distributed along the plasma membrane at 3, 6, 12, and 24 hr after inoculation. However, within 12 hr after bacterial challenge in resistant interactions, PLD was clustered preferentially in membranes adjacent to bacterial cells.     

Growth Inhibition of Helicobacter pylori by Monoclonal Antibody to Heat-Shock Protein 60

The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth. The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.     

Comparative in vitro and ex vivo studies on the bactericidal activity of Tetraclean, a new generation endodontic irrigant, and sodium hypochlorite

The aim of this study was to compare the efficacy of a new generation endodontic irrigant, Tetraclean, to the widely used sodium hypochlorite. Tetraclean combines a powerful detergent effect with a strong antimicrobial efficacy, whereas sodium hypochlorite has several drawbacks and is sometimes ineffective in preventing microbial-mediated endodontic failure. The bactericidal activity of both irrigants against Enterococcus faecalis, the most commonly isolated species from root canals of teeth with post-treatment disease, was assessed i) in vitro, according to the European Standard lines for the evaluation of the bactericidal activity of chemical disinfectants, and ii) with an ex vivo model of extracted and decoronated human teeth, infected with E. faecalis and subsequently irrigated with either of the irrigants. Both irrigants display very similar bactericidal activity against E. faecalis in vitro. However, the ex vivo model shows that only in the teeth irrigated with Tetraclean did the bacterial burden gradually drop until no bacteria were detectable a few days post-irrigation. Vice versa, in the teeth irrigated with sodium hypochlorite, the drop in the bacterial burden was rapid but temporary and most of the teeth were colonized again by 48 hours post-irrigation.     

Virus DNA replication and protein synthesis in Amsacta moorei entomopoxvirus-infected Estigmene acrea cells

Amsacta moorei entomopoxvirus DNA synthesis was detected in Estigmene acrea cells by [³H]thymidine incorporation 12 hr after virus inoculation. Hybridization of ³²P-labeled Amsacta entomopoxvirus DNA to the DNA from virus-infected cells indicated that viral-specific DNA synthesis was initiated between 6 and 12 hr after virus inoculation. A rapid increase in the rate of virus DNA synthesis was detected from 12 to 24 hr after virus inoculation. Amsacta entomopoxvirus protein biosynthesis in E. acrea cells was studied by [su35S]methionine incorporation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extracellular virus and virus-containing occlusion bodies were first detected in virus-infected cell cultures 18 hr after virus inoculation. Thirty-seven virus structural proteins, ranging in molecular weight from 13,000 to 208,000 were detected in both occluded and nonoccluded forms of the virus. The biosynthesis of virus structural proteins increased rapidly from 18 to 34 hr after infection. A major viral-induced protein corresponding in molecular weight to viral occlusion body protein (110,000) was detected approximately 24 hr after virus inoculation.     

Studies on the in vivo cellular reactions of insects: an ultrastructural analysis of nodule formation in Galleria mellonella.

Cellular aggregates or nodules which formed in Galleria mellonella larvae in response to injections of killed Bacillus cereus were examined during their first 24 hr of development. The initial aggregation stage was very rapid, occurring within 1 min, and was brought about by the release of an adhesive flocculent material by granular haemocytes in contact with the bacteria. This material, which subsequently became increasingly electron dense owing to melanin deposition, consolidated and by 5 min ramified all the intercellular spaces. The granular cells degenerated, releasing portions of cytoplasm form their peripheries, and eventurally appeared in the matrix as voids containing loosely flocculent material. No bacterial breakdown was seen, however. From 2-4 hr, plasmatocytes began to encapsulate the necrotic mass, and by 24 hr a complete sheath, composed of three discrete regions of cells, was formed. These findings are discussed in relation to the various proposed mechanisms of nodule and capsule formation.     

SOME OBSERVATIONS ON BACTERICIDAL PAINT FILMS

SUMMARY: A method is described for testing the self-sterilizing activity of surfaces coated with paints containing bactericidal agents. The most active coating consisted of 10% (w/w) Cetavlon dissolved directly in urea-formaldehyde:alkyd resin. The recovery of viable organisms from such surfaces infected with Streptococcus faecalis, Staphylococcus aureus, Aerobacter aerogenes and Chromobacterium prodigiosum was less than 10% of that from painted control surfaces after exposure for 1 hr at atmospheric relative humidity of 70% and temperature of 20°. Bacillus subtilis spores were not affected until they became heat-sensitive. The self-sterilizing activity was markedly diminished at 40% r.h. and the inclusion of blood in the deposited bacterial particle was then completely protective. Repeated washing in water with gentle rubbing quickly inactivated Cetavlon paint films.     

烟青虫核型多角体病毒的复制和染病后血淋巴蛋白的变化

用烟青虫(Hiliothis assulta)核型多角体病毒感染5龄初烟青虫幼虫,以染病后24、48·,72、96/120小时分别测定了虫体血淋巴蛋白浓度的变化。幼虫染病后24小时血淋巴蛋白浓度要高于同期对照组,72小时后染病幼虫血淋巴蛋白浓度较对照急剧下降。在染病及对照血淋巴样品中电泳分析(PAGE)三种蛋白(普通蛋白、糖蛋白和脂蛋白)均可分别染出22条、3条和3条带。分析结果表明,在虫体正常生长代谢过程中发生变化的主要蛋白可能大多为糖脂复合蛋白,但病毒的侵染能抑制这些变化的产生。电镜观察染病毒后幼虫的中肠组织和气管上皮组织,发现中肠染病轻微,不形成多角体。气管上皮细胞感染情况表明其属于对NPV感染较为敏感的组织之一,并在虫体染病后的病毒二次感染上可能起着重要作用。     

Comparative analysis of calf and cow oocytes during in vitro maturation

To determine possible causes of reported differences between developmental competence of oocytes isolated from prepubertal (10- to 14-week-old calves) and adult cows, three parameters were analysed, comparatively, during in vitro maturation (IVM): (1) oocyte diameter, (2) oocyte energy metabolism, and (3) protein synthesis of oocytes and cumulus cells. Cumulus-oocyte complexes were isolated from follicles of 3–5 mm in diameter in both age groups. Mean oocyte diameter was smaller (P < 0.02) in calves than in cows (118.04 ± 1.15 versus 122.83 ± 0.74 μm). During the first 3 hr of IVM, calf oocytes metabolised glutamine and pyruvate at lower rates than adult oocytes, but after 24 hr of culture, both molecules were metabolised at the same rate as for adult oocytes. A significant decrease in protein synthesis, as measured by [³⁵S]methionine and [³⁵S]cysteine incorporation was recorded after 9 hr of IVM in calf oocytes, while in adult oocytes a significant decrease in protein synthesis was detected only after 24 hr. After the first 3 hr of maturation, proteins of 130, 26, and 24 kDa were more abundant in adult than in calf oocytes, while a protein of 55 kDa was more visible in calf than in adult oocytes. At the same time, among proteins newly synthesised by cumulus cells, molecules of 405, 146, 101, and 77 kDa were more abundant in adults than in calves. In conclusion, calf oocytes and cumulus cells showed several differences when compared with their adult counterparts, which are consistent with their reported lower developmental competence. Mol. Reprod. Dev. 49:168–175, 1998. © 1998 Wiley-Liss, Inc.     

Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication

The titer of Amsacta entomopoxvirus (EPV) protein detected in murine L-929 cells by enzyme-linked immunosorbent assay (ELISA) decreased to within preimmune serum levels by 24 hr after inoculation of the virus which indicates that Amsacta EPV structural protein biosynthesis does not occur in the vertebrate cell line. A viral-induced protein of approximately 100,000 M_r was detected by [³⁵S]methionine incorporation 4 hr after inoculation of Tn-368 cells with Amsacta EPV. Biosynthesis of protein which reacted with vaccina antiserum was detected in Estigmene acrea (BTI-EAA) cells by ELISA 10 hr after inoculation with 10 PFU of virus per cell. The amount of putative vaccinia structural protein detected in BTI-EAA cells increased approximately twofold by 70 hr after virus inoculation. No increase in vaccinia structural protein biosynthesis was detected in BTI-EAA cells inoculated with vaccinia virus previously inactivated by heat and UV light.     

Overexpression of Mouse Estrogen Receptor-β Decreases but Its Transactivation and Ligand Binding Domains Increase the Growth Characteristics of <Emphasis Type="Italic">E. coli</Emphasis>

Escherichia coli is one of the most common and widely used prokaryotic hosts for the expression of recombinant proteins. The overexpression of recombinant proteins occasionally increases bacterial growth but sometimes reduces it and becomes lethal to the host cells. Here, we report the overexpression of mouse ER-β and its domains in the prokaryotic expression system and its opposite effect on the growth characteristics of E. coli. ER-β protein was immunologically detected as a 53 kDa his-tag protein in the pellet of the bacterial lysate. Its overexpression, as reflected by the total protein content and expression pattern, resulted in the decrease of bacterial growth. However, the overexpression of ER-β transactivation domain (TAD) using pIVEX and ligand binding domain (LBD) using pRSETA in E. coli BL21 (DE3) show opposite pattern. TAD was immunologically detected as 20 kDa and LBD as 22 kDa protein in the supernatant of the bacterial lysate and their overexpression increased the bacterial growth.     

The reduction of Fusobacterium nucleatum in mice is irrelevant to the nitric oxide induced by iNOS

Previously we reported that mice infected recurrently with live Fusobacterim nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after the Fn injection. We now investigated whether the NO has the capability of killing Fn, a gram-negative rod periodontal pathogen. The mice were divided into three groups: treated with live bacteria (LB), treated with heat-killed bacteria (HKB) and untreated: normal (N). The Fn reduction, NO production and cell number after Fn injection were then compared in these mice. In the LB group, no Fn was detected at 6 hr, whereas it was still detected in the HKB and N groups at 24 hr as assessed by both colony counts and PCR assays. A significant amount of NO was synthesized in the LB group at 24 hr after the Fn injection. Fn is not killed by SNAP-generated NO in vitro. An increase in the total cell number was accompanied by an increase of the neutrophil numbers in the LB group. Intracellular O2(-) generation (including ONOO(-)) was visualized using dihydrorhodamine (DHR)-123. The peak of O2(-) generation by PEC was shown to be at 3 hr in all 3 groups. The number of O2(-) positive cells in the LB group at 3 hr was remarkably high, and most of them were likely to be neutrophils. The Fn reduction would be performed cooperatively via oxygen dependent and oxygen independent mechanisms. Thus reactive oxygen species (ROS) included in the oxygen dependent mechanism appear to be important for Fn reduction. However the significant amounts of NO derived from the iNOS synthesized in the LB group between 12 hr and 24 hr after injection of LFn were not involved in the Fn reduction.     

Induction of Prodigiosin Biosynthesis after Shift-Down in Temperature of Nonproliferating Cells of Serratia marcescens

Nonpigmented bacteria obtained by growth of Serratia marcescens at 38 C synthesized prodigiosin at 25 C if certain individual amino acids were added to cultures of nonproliferating cells. In order of effectiveness, the amino acids were: DL-histidine, L-proline, L-hydroxyproline, DL-alanine, L-alanine, DL-aspartic acid, D-alanine, DL-proline, L-serine, L-ornithine, L-glutamic acid, and D-proline. DL-Histidine at its optimal concentration (20 mg/ml) induced formation of prodigiosin (198 mug of prodigiosin per mg of bacterial protein) after incubation of cultures for 54 hr. Lower concentrations (10 mg/ml) of the other amino acids usually were optimum but less prodigiosin was synthesized, and the maximal amount of pigment occurred between 36 and 48 hr. DL-Methionine was not effective alone but at a low concentration (40 mug/ml) enhanced and accelerated biosynthesis of prodigiosin in the presence of other suitable amino acids. Addition of 2 mg of L-proline per ml at 0 hr induced formation of only 30 mug of prodigiosin after incubation for 42 hr, but addition at 36 hr of 5 mg more of L-proline per ml increased synthesis to 120 mug at 42 hr. Again, DL-methionine markedly augmented prodigiosin biosynthesis in these cultures. Synthesis of prodigiosin ceased if cultures were shifted from 25 to 38 C. Prodigiosin biosynthesis by the nonproliferating cells was maximum when cultures were aerated, the amount of bacterial protein was about 2.0 mg/ml, and amino acids were added at 0 hr. Bacteria synthesized prodigiosin most efficiently when they were harvested from aerated cultures grown at 38 C for 24 hr in a complete medium in a fermentor.     

Improved adsorption of an Enterococcus faecalis bacteriophage ΦEF24C with a spontaneous point mutation

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component.     

Overproduction of mouse estrogen receptor alpha-ligand binding domain decreases bacterial growth

Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse estrogen receptor alpha (mouse ERα) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERα-LBD, as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth.     

Measles Virus Ribonucleic Acid and Protein Synthesis: Effects of 6-Azauridine and Cycloheximide on Viral Replication

Cycloheximide and 6-azauridine were employed to study the time course of measles virus protein and nucleic acid syntheses in AV3 cells. Synthesis of ribonucleic acid (RNA) essential for infectivity was first detected at 6 hr and increased concurrently with the formation of essential protein. Maximum levels of virus-specific RNA and protein were present by 18 hr, a time when only 5% of progeny virus was detected. Essential RNA and protein syntheses preceded the formation of infectious virus by at least 10 to 12 hr. The time course of RNA and protein syntheses essential for the formation of complement-fixing (CF) antigen and salt-dependent agglutinin (SDA) was also determined. RNA synthesis essential for the formation of SDA was first detected at 2 hr and was present maximally by 6 hr, whereas SDA-protein increased concurrently with the protein essential for infectivity. This suggested that the last protein essential for infectivity may be SDA. RNA synthesis essential for the formation of CF antigen was first detected at 4 hr, while CF-protein increased at 5 hr and preceded SDA-protein and protein essential for infectivity by approximately 3 hr. Reversal of inhibition of protein synthesis by cycloheximide indicated that early protein synthesis (1 to 3 hr) was required for the formation of infectious virus. The data suggest that the relatively long eclipse period observed with measles virus is related to a long maturation period rather than to late formation of early proteins, viral RNA, or structural proteins.     

Pulmonary hypertension syndrome in young chickens challenged with frozen and autoclaved cultures of Enterococcus faecalis

Enterococcus faecalis, when administered in a growth medium or sterile saline, will cause pulmonary hypertension syndrome (PHS) in chickens. The objective of this study was to determine if frozen and/or autoclaved cultures of E. faecalis retain ability to evoke PHS. In Trial 1, chicks were inoculated with 3.6 x 10(7) E. faecalis (IA) in tryptic soy broth (TSB) from either a live culture or one that had been autoclaved (120 degrees C for 20 min). Controls received TSB. Autoclaved and live cultures produced the same degree of PHS in a majority of the birds. Trial 2 used the same protocol, except a frozen (-70 degrees C for 60 min) culture of E. faecalis was compared with the control. The results agreed with those of Trial 1, i.e., the frozen culture also produced PHS. Trial 3 was conducted to determine if E. faecalis caused PHS by producing and releasing some unknown substance into the supernatant. Incidence of PHS was based on percentage of birds exhibiting ascites fluid at 24 hr after challenge. Controls received sterile, frozen, or autoclaved TSB. As compared with controls, those birds that received challenge with E. faecalis alone, supernatant alone, and E. faecalis plus supernatant from live cultures exhibited similar incidence of ascites, whereas birds that received E. faecalis plus supernatant and supernatant alone from cultures that had been either frozen or autoclaved exhibited elevated incidence of ascites as compared with controls. Also, with frozen and autoclaved cultures, those birds that received only pelleted E. faecalis exhibited incidence of ascites that did not differ from controls. Apparently, E. faecalis produces PHS in chicks by producing and releasing an unknown toxin.