Identification of a new enterotoxin as enterotoxin C

Bergdoll, Merlin S. (University of Chicago, Chicago, Ill.), Concordia R. Borja, and Remedios M. Avena. Identification of a new enterotoxin as enterotoxin C. J. Bacteriol. 90:1481-1485. 1965.-Identification of a new enterotoxin was accomplished by purification of the enterotoxins produced by staphylococcal strains 137 and 361 and by the preparation of specific antitoxin to the enterotoxin. Toxicity of the preparations was determined in rhesus monkeys, and specificity of the enterotoxin-antitoxin reaction was determined in gel-diffusion plates. The enterotoxin has been designated enterotoxin C, and staphylococcal strain 137 (ATCC 19095) was selected as the prototype strain.     

Biologic Activity of Cell-Associated Staphylococcal Enterotoxin A

Cell associated staphylococcal enterotoxin A, released by lysostaphin treatment of Staphylococcus aureus cells, was found to be biologically active in cynomolgus monkeys. The activity was comparable to that of extracellular enterotoxin A; six of six monkeys vomited within 5 h in response to extracellular enterotoxin and four of six also vomited in response to the same serologic level (4.8 μg per monkey) of cell-associated enterotoxin. Feeding S. aureus cells containing cell-associated enterotoxin A to cynomolgus monkeys resulted in emesis in three of five monkeys within 3 h. This suggests that consumption of S. aureus cells could lead to staphylococcal intoxication.     

Leukocyte mobilization induced by hypervolemia is due to a combined alpha- and beta-adrenoceptor activation

A phenomenon of leukocytosis induced by hypervolemic stress was discovered. Although a single injection of 350 microl of saline (equivalent to approx. 70 ml in humans, 1 ml/kg of body weight) did not have an effect on the leukocyte counts in long-term intravenously cannulated, freely behaving rats, a single injection of 750 microl of saline (equivalent to approx. 150 ml in humans, 2.1 ml/kg) induced rapid leukocytosis of 160% within 1 minute followed by a gradual increase up to 180% after 1 hour. Measurement of serum norepinephrine concentration revealed a significant increase in rats of the hypervolemic group, compared with those of the low volume group. Pretreatment with either the beta-adrenoceptor antagonist nadolol or the selective alpha2-adrenoceptor antagonist yohimbine prevented both leukocyte peaks in the high volume group, suggesting a combined receptor activation. This critical dependence of leukocyte counts on changes in blood volume should be taken into consideration in experiments with laboratory animals (the quantity of volume applications can falsify results of experiments).     

The Rhesus monkey as a model for the study of infectious disease

Models for bacterial and viral infections and intoxication were developed in rhesus macaques (Macaca mulatta). Manifestations of acute-phase illnesses, e.g., temperature, white blood cell (WBC) counts, blood cultures, etc., were monitored at regular intervals. Viral infection was established by inoculating subcutaneously 412 plaque-forming units of Trinidad strain, Venezuelan equine encephalomyelitis virus. A diphasic febrile response developed, with the first fever peak on days 1 to 2 and a second peak on days 3 to 5. Viremia occurred within 12 hours and persisted in some animals for as long as five days. WBC responses were typical of viral infection. Gram-positive infections were induced by intravenous (IV) inoculation of 10⁸ Type I Diplococcus pneumoniae. Peak febrile response and bacteremia (10² to 10⁶ pneumococci per milliliter) occurred within 48 hours. Gram-negative infections, obtained by IV inoculation with 10⁹ Salmonella typhimurium, induced maximal febrile responses within 24 to 48 hours. Leukopenia occurred in 75% of animals; all were bacteremic. Mortality was 40% at 72 hours. Manifestations of intoxication following IV administration of purified staphylococcal enterotoxin B (10 μg per kg body weight) consisted of vomiting, diarrhea, leukopenia, and fever within one to three hours and resembled nonlethal staphylococcal food poisoning of man. These studies indicate that the rhesus macaque has reproducible and characteristic responses to a variety of microbial stimuli and therefore is eminently suitable for studying pathophysiologic, metabolic, and immunologic parameters of infectious or toxic disease processes.     

Role of the Kidney in Staphylococcal Enterotoxemia

Highly purified staphylococcal enterotoxin B (SEB) is known to accumulate rapidly within the kidneys of experimental animals. The present study was performed to determine whether the predominant renal localization of SEB was of fundamental pathophysiologic importance in the development of lethal shock after the intravenous administration of this toxin to monkeys. Eight bilaterally nephrectomized Macaca mulatta given 10 mug of SEB per kg survived for an average time period less than half that of nephrectomized control animals (P < 0.001). Their survival time, however, was similar to that of control, sham-nephrectomized monkeys given an equal amount of SEB. Thus, no evidence was obtained to suggest that the kidney converted purified SEB to a more potent toxin. The glomerular filtration and proximal tubule cell accumulation of SEB possibly occurred as a nonspecific consequence of its molecular size, and such localization within the kidney might have served to reduce the quantity of SEB reaching some other site of toxic activity. Similar pathological and clinical findings were demonstrated in monkeys from both experimental and control groups; these could not be ascribed to SEB alone.     

Effect of pH and Oxygen Tension on Staphylococcal Growth and Enterotoxin Formation in Fermented Sausage

Commercial fermented 0sausages that contained significant numbers of viable coagulase-positive staphylococci were found to have the growth localized in the outermost areas of the sausage where oxygen tension was highest. Staphylococci were found to be more acid-tolerant aerobically than anaerobically. With chemical acidulation of sausage, growth could be controlled both aerobically and anaerobically with approximately 1.5% glucono delta lactone. Biological acidulation with a high inoculum of Pediococcus cerevisiae inhibited anaerobic staphylococcal growth but failed to suppress aerobic growth completely. A staphylococcal count of approximately 4 × 10⁷ cells/g of sausage appeared to be necessary to produce detectable enterotoxin A within 24 hr in sausage. A minor difference existed in the relative rates of production of the different types of enterotoxin. Detectable enterotoxin A was produced in 24 hr in sausage held in atmospheres containing 10, 15, and 20% oxygen. In an atmosphere containing 5% oxygen, toxin was detected after 48 hr of incubation. No toxin was detected after 120 hr under anaerobic conditions. Most staphylococcal strains tested initiated growth and produced detectable enterotoxin aerobically at a pH of 5.1 in broth media. Anaerobically, however, most strains failed to produce detectable enterotoxin below pH 5.7.     

Effect of Staphylococcal Enterotoxin B on the Electroencephalogram of Monkeys

A highly purified preparation of staphylococcal enterotoxin B was administered intravenously, 1 mg/kg, to rhesus monkeys. Electroencephalograms (EEG) were recorded from electrodes attached to the skin or implanted on the dura. The dose of toxin employed consistently produced a sequence of vascular collapse followed by death; in control studies, animals were bled periodically to produce a similar pattern of shock. Regardless of the time to death following administration of the enterotoxin, there were essentially no changes from base line EEG patterns until shortly before death. With the development of preterminal severe shock, there was a marked decrease in EEG wave frequency and an initial increase in amplitude. The latter diminished progressively to produce an isoelectric tracing immediately prior to death. This could be reversed for a brief period by epinephrine. An identical sequence of EEG changes was observed during the terminal period of hemorrhagic shock. It is postulated that cerebral anoxia, caused by inadequate blood flow, is the primary cause of the altered EEG patterns that accompany enterotoxin toxicity. In this respect, staphylococcal enterotoxin B produces changes apparently similar to bacterial endotoxin but distinctly different from the EEG effects reported after botulinum toxin, anthrax toxin, or rattlesnake and cobra venom.     

Megaloblastic anemia in patients receiving total parenteral nutrition without folic acid or vitamin B12 supplementation.

Pancytopenia developed in four patients receiving postoperatively total parenteral nutrition (TPN). Symptoms and signs were related mainly to underlying bowel disease. Hematologic abnormalities, first noted from 4 to 7 weeks following institution of TPN, consisted of normocytic anemia (mean decrease in hemoglobin value, 2.2 g/dL), occasional macrocytes being noted, leukopenia (range of leukocyte counts, 1.2 to 3.6 X 10(9) L), some hypersegmented neutrophils being detected, and clinically significant thrombocytopenia (range of platelet counts, 25 to 52 X 10(9)/L). In all patients the bone marrow showed megaloblastic changes, with ring sideroblasts, although pyridoxine was included in the TPN regimens. Serum vitamin B12 values were normal in one patient and at the lower limit of normal in the other two patients in whom it was measured, while serum or erythrocyte folate values, or both, were reduced in three patients. Full hematologic response was observed in the four patients after folic acid replacement therapy; leukocytosis and thrombocytosis were noted in three. Thus, folic acid and possibly vitamin B12 should be added routinely to TPN regimens to prevent deficiency of either substance.     

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1.

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.     

Chromatofocusing: a new method for purification of staphylococcal enterotoxins B and C1

A new chromatographic procedure was developed which obtained highly purified preparations of staphylococcal enterotoxins B and C1 in yields of 60% from cultures of Staphylococcus aureus and which is faster than any of the separation methods used previously. The procedure involves chromatography on carboxymethylcellulose, removal of alpha-toxin by adsorption to rabbit erythrocyte membranes, and finally, chromatofocusing as the fundamental new step. Enterotoxins were obtained in highly purified form and behaved in a homogeneous manner as determined by ultracentrifugation and electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, with molecular weights of 34,000 for staphylococcal enterotoxin B and 30,000 for staphylococcal enterotoxin C1. Using chromatofocusing as the final purification step, we isolated three B and six C1 distinct but immunologically identical enterotoxin fractions, which were found to be devoid of any impurities and to possess a marked degree of toxicity in monkeys.     

Enterotoxin B: Serological Assay in Cultures by Passive Hemagglutination

Bis-diazotized benzidine hemagglutination with Formalinized sheep erythrocytes was adapted to the rapid and specific detection of enterotoxin B in staphylococcal culture fluids. There was complete agreement between hemagglutination inhibition (HI) and gel diffusion in detecting 8 enterotoxin B-positive cultures from a total of 68 staphylococcal cultures tested. The sensitivity of HI equals or exceeds that of gel diffusion. Also, results can be obtained in several hours, even with extremely low concentrations of enterotoxin, whereas it may require 24 hr to 1 week to obtain comparable results with gel diffusion. Problems associated with the presence of potent hemagglutinins for sheep erythrocytes in several staphylococcal culture fluids are discussed.     

Naturally occurring Yersinia enterocolitica septicemia in patas monkeys (Erythrocebus patas)

Two cases of Yersinia enterocolitica septicemia occurred in a breeding group of 22 adult patas monkeys (Erythrocebus patas). Affected animals had acute clinical signs of depression, weakness, dehydration, hypothermia, hepatomegaly and pronounced leukopenia. Both animals died a few hours after treatment was initiated. Gross necropsy findings included jaundice, fluid in body cavities, hepatomegaly, splenomegaly, multiple white foci within the liver and spleen, generalized lymph node enlargement and numerous mucosal ulcerations in the colon. Primary histopathological lesions were multifocal hepatic necrosis, splenic necrosis, chronic ulcerative enteritis and diaphragmatic myositis with necrosis and edema. Yersinia enterocolitica was cultured from the liver, spleen, lung, jejunum and rectum. Wild rodents, particularly mice, may have been a source of infection for these animals, as the monkeys were housed in a rural, indoor-outdoor facility. A preliminary culture survey showed that some clinically normal patas monkeys harbored the organism in their intestinal tracts.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E.

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Specificity and cross-reactivity of staphylococcal enterotoxin A monoclonal antibodies with enterotoxins B, C1, D, and E

The cross-reactivity of monoclonal antibodies produced against staphylococcal enterotoxin A with purified and crude enterotoxins B, C1, D, and E and the specificity of such reactions were evaluated by the indirect enzyme-linked immunosorbent assay and immunoblotting of Western blots (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) followed by autoradiography. Purified and crude enterotoxins B were also tested with polyclonal antibodies. Specificity of reactivity was demonstrated by immunoblotting of crude enterotoxin A, crude enterotoxin A treated with trypsin, crude enterotoxin E, and also with crude A, B, C1, and D that were pretreated with Sepharose-4B-linked normal rabbit immunoglobulin G to remove protein A. A band corresponding to each staphylococcal enterotoxin was seen with monoclonal antibodies under all conditions tested and also with crude and purified enterotoxin B with two different (rabbit and goat) polyclonal antisera.     

Bone marrow hypoplasia associated with estrus in ferrets

Bone marrow hypoplasia was characterized in a group of female ferrets during prolonged estrus. All ferrets exhibited hematological changes characteristic of various degrees of bone marrow hypoplasia. Hematological findings included initial thrombocytosis and leukocytosis followed by thrombocytopenia, leukopenia and anemia. Platelet counts below 50,000/microliters were observed in 55% of the ferrets. Hemorrhagic anemia due to thrombocytopenia was the most common cause of death and the mortality rate was 40%. Histopathological findings included bone marrow hypoplasia affecting all cell lines and decreased splenic extramedullary hematopoiesis.     

Clinical Chemistry of Staphylococcal Enterotoxin Poisoning in Monkeys

Clinical chemistry values were examined in 90 monkeys administered a purified preparation of staphylococcal enterotoxin, type B, intravenously. These studies showed an early release of epinephrine accompanied by a mild increase in blood glucose. This was followed by progressively developing prolonged hypoglycemia. An early increase in bloodurea nitrogen occurred, presumably as a result of both prerenal azotemia and functional renal failure seen in association with the observed hypotension. Serum protein, Ca, and Cl concentrations decreased with time. Pi levels increased, whereas Na and K concentrations in serum remained unchanged. Serum enzyme concentrations were unchanged, with the exception of serum glutamic oxaloacetic transaminase, which rose rapidly when compared with prechallenge control observations or with values from sham-challenged monkeys. These changes were statistically significant. These results suggested that enterotoxin administered intravenously produced early change in glucose metabolism, possibly related initially to catecholamine release and later to increased utilization of glucose and metabolic acidosis. Other findings were compatible with tissue breakdown at as yet undetermined locations and with loss of endothelial membrane integrity, as evidenced by loss of protein from the vascular space.     

Capillary Tube Immunological Assay for Staphylococcal Enterotoxins

A simple assay is reported in which 1 mug of staphylococcal enterotoxins A, B, and D per ml was detected in less than 1 hr. Interfacial reaction of antisera and enterotoxin solutions in a 1-ml internal diameter capillary tube allowed rapid detection of sera type.     

Plasma macrophage colony-stimulating factor levels during cardiopulmonary bypass with extracorporeal circulation

Leukocytosis and thrombocytopenia occur during cardiopulmonary bypass (CPB) with extracorporeal circulation (ECC). Elevated circulating concentrations of macrophage colony-stimulating factor (M-CSF) are reported during thrombocytopenia and leukopenia of different origins. We have assessed M-CSF concentrations in 40 patients undergoing CPB with ECC. Plasma M-CSF concentrations were stable during ECC and increased at the 6th (7.3 +/- 0.7 IU/mug protein) and 24th (8.6 +/- 0.8 IU/mug protein) postoperative hour compared with pre-ECC values (4.9 +/- 0.5 IU/mug protein). A deep thrombocytopenia was found during ECC and until the 24th postoperative hour. A drop of leukocyte counts was found during ECC followed by an increase after ECC weaning. While no correlation was found between M-CSF concentrations and the leukocyte counts, M-CSF values were positively correlated with platelet counts only before and during ECC. Thus, M-CSF is not implicated in the thrombocytopenia and the leukopenia generated during CPB with ECC. However the elevated levels of M-CSFa few hours after the end of ECC might play a role in the inflammatory process often observed after CPB.     

Changes in the Differential Leukocyte Count of Chicks Inoculated with Salmonella

Studies were conducted to determine the effects of Salmonella anatum and S. heidelberg infections on the differential leukocyte counts of baby chickens. Newly hatched broiler-type chicks were inoculated in the yolk sac with suspensions of either S. anatum or S. heidelberg. At 0, 24, 48, and 72 hr after inoculation, blood was taken by heart puncture from chicks of both inoculated groups and from a group of uninoculated chicks. The averages of the leukocyte counts of three or four chicks from each group were used as the blood values for specific time intervals. The six classes of leukocytes counted were lymphocytes, monocytes, juveniles, heterophils, eosinophils, and basophils. The leukocytes classified as juveniles were immature or degenerate heterophils and were found almost exclusively in the infected chicks. Changes in heterophil, juvenile, and lymphocyte counts were affected by both the number of cells in the inoculum (300 versus 3 million cells of S. anatum) and species (S. anatum and S. heidelberg). Infection with either Salmonella species resulted in the development of a severe heterophilic leukopenia, and a significant increase in the percentage of both juveniles and lymphocytes by 48 hr postinoculation. Mortality rate was higher in groups of chicks inoculated with S. heidelberg than in groups given S. anatum.     

Nucleotide sequence of the type A staphylococcal enterotoxin gene.

We determined the nucleotide sequence of the gene encoding staphylococcal enterotoxin A (entA). The gene, composed of 771 base pairs, encodes an enterotoxin A precursor of 257 amino acid residues. A 24-residue N-terminal hydrophobic leader sequence is apparently processed, yielding the mature form of staphylococcal enterotoxin A (Mr, 27,100). Mature enterotoxin A has 82, 72, 74, and 34 amino acid residues in common with staphylococcal enterotoxins B and C1, type A streptococcal exotoxin, and toxic shock syndrome toxin 1, respectively. This level of homology was determined to be significant based on the results of computer analysis and biological considerations. DNA sequence homology between the entA gene and genes encoding other types of staphylococcal enterotoxins was examined by DNA-DNA hybridization analysis with probes derived from the entA gene. A 624-base-pair DNA probe that represented an internal fragment of the entA gene hybridized well to DNA isolated from EntE+ strains and some EntA+ strains. In contrast, a 17-base oligonucleotide probe that encoded a peptide conserved among staphylococcal enterotoxins A, B, and C1 hybridized well to DNA isolated from EntA+, EntB+, EntC1+, and EntD+ strains. These hybridization results indicate that considerable sequence divergence has occurred within this family of exotoxins.