The neutrophil chemiluminescence response to Pneumocystis carinii is stimulated by GM-CSF and gamma interferon

An enzyme-linked immunosorbent assay (ELISA) with antibodies specific to beta, epsilon and iota ib toxins of Clostridium perfringens was developed to detect beta, epsilon and iota ib toxins, respectively. The ELISA was sensitive enough to detect as little as 1.0 ng/ml of purified beta and iota ib toxins and 0.1 ng/ml of purified epsilon toxin. By means of the ELISA method, 192 isolates of C. perfringens from food samples in Japan and Thailand, and 58 isolates from patients suffering from gas gangrene or gastroenteritis were examined. One isolate from food samples in Japan, three from food samples in Thailand and five from stools of patients with gastroenteritis were C. perfringens type D. One type B and one type C were detected from the stools of patients with gastroenteritis.     

The production and evaluation of monoclonal antibodies to Clostridium perfringens type D epsilon toxin

The production of five monoclonal antibodies to the epsilon prototoxin of Clostridium perfringens is described. All five monoclonal antibodies located three proteins in a trypsinized preparation of epsilon prototoxin. These proteins were located at 37.6 kDal, 35.6 kDal and 33.7 kDal by Western blots. Two of the monoclonal antibodies, M26/2 and M27/12, neutralized epsilon toxin in the mouse lethality assay. Four of the five monoclonal antibodies are considered suitable as reagents to detect epsilon toxin in assay procedures.     

In vitro tests for the measurement of clostridial toxins, toxoids and antisera. II. Titration of Clostridium perfringens toxins and antitoxins in cell culture

The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.     

Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia

Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia.     

Diagnosis of Clostridium perfringens intestinal infections in sheep and goats

Clostridium perfringens produces disease in sheep, goats and other animal species, most of which are generically called enterotoxemias. This micro-organism can be a normal inhabitant of the intestine of most animal species including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates in large numbers and produces several potent toxins that are absorbed into the general circulation or act locally with usually devastating effects on the host. History, clinical signs and gross post-mortem findings are useful tools for establishing a presumptive diagnosis of enterotoxaemia by C. perfringens in sheep and goats, although no definitive diagnosis of these diseases can be made without laboratory confirmation. Because all types of C. perfringens can be normal inhabitants of the intestine of most animals, culture of this micro-organism from intestinal contents of animals has no diagnostic value unless a colony count is performed and large numbers (usually more than 10(4)-10(7)CFU/g) of C. perfringens are found. The most accepted criterion in establishing a definitive diagnosis of enterotoxaemia by C. perfringens is the detection of its toxins in intestinal contents. However, some of the major toxins of C. perfringens (i.e. epsilon toxin) can also be found, albeit in small amounts, in the small intestine of clinically normal sheep, and this poses a diagnostic challenge. In such cases the histopathology of the brain must be used as an alternative diagnostic tool, since the lesions produced by epsilon toxin in the brains of sheep and goats are unique and pathognomonic for C. perfringens type D enterotoxaemia. Ancillary tests, such as measurement of urine glucose or observation of Gram stained smears of intestinal mucosa can be used and, although they have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxaemia if they are negative. In conclusion, the diagnosis of C. perfringens infections in animals is complex and it is appropriate to rely on a combination of diagnostic techniques rather than one singe test.     

Comparison of the nucleotide sequence and development of a PCR test for the epsilon toxin gene of Clostridium perfringens type B and type D

The sequence of the epsilon toxin gene of Clostridium perfringens type D was determined and compared with that of the previously reported type B sequence. It showed two nucleotide changes in the open reading frame, giving rise to one amino acid substitution. The promoter sequences were not homologous, and different putative -35 and -10 regions have been identified in each. The sequence information was used to develop PCR primers which were specific for the epsilon toxin gene. The utility of this system for identifying type B or D strains of C. perfringens was demonstrated.     

PCR detection of Clostridium perfringens type D in formalin-fixed, paraffin-embedded tissues of goats and sheep

A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep. When pure cultures of Cl. perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers). When used to identify Cl. perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl. perfringens. No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique.     

Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin

Abstract A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay ( r = 0.991) and over a wide range of viability (15–75%) as determined by ethidium bromide/acridine orange staining ( r = 0.995).     

An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon components of multivalent sheep vaccines

Potency testing of veterinary vaccines containing clostridial antigens currently requires the vaccination of laboratory rabbits followed by the determination of specific antitoxin concentration in the rabbit sera by toxin neutralization test in mice. ELISAs are described as an alternative method to toxin neutralization for the determination of Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins. The assays were found to be rapid, specific and economical and showed good correlation with the toxin neutralization test.     

An investigation of the prevalence of the toxigenic types of Clostridium perfringens in horses with anterior enteritis: preliminary results

Equine anterior enteritis is an acute syndrome with unknown aetiology, although salmonellosis and infection with Clostridium perfringens have both been suggested as potential causes. The main aim of this preliminary study was to compare the prevalence of toxigenic types of C. perfringens in clinically healthy horses and in horses with anterior enteritis. From horses admitted with colic at Phillip Leverhulme Large Animal Hospital in 1995-1996, samples of gastric reflux, small intestinal contents and faeces were taken for isolation of C. perfringens. Five of those horses were admitted as anterior enteritis cases, of which C. perfringens was isolated in pure culture in all five horses. Two of the anterior enteritis cases from which viable bacterial counts had been performed revealed 10(6) CFU/g faeces C. perfringens. Samples of gastric reflux and small intestinal contents submitted from one of these horses revealed 10(4) CFU/mL and 10(5) CFU/mL respectively. The number of C. perfringens observed in the gastric reflux was considered significant as the total volume removed was 12 L. The counts observed in faeces taken from horses admitted with anterior enteritis, were significantly higher than the <10(2) CFU/g faeces observed in faeces from healthy horses and horse presenting with colic and with other diagnoses. The major toxigenic types of C. perfringens in both healthy and diseased horses are being investigated using the polymerase chain reaction (PCR) to amplify target DNA sequences of the toxin genes. Primers have been designed from the published DNA sequences of the enterotoxin, alpha, beta, epsilon and iota toxin genes. PCR products obtained from NCTC strains of C. perfringens have been cloned and the sequenced, to verify that the amplicon sequence is correct. Initial typing suggests that C. perfringens type A is the predominant toxin type isolated from healthy horses and horses with colic with other diagnoses.C. perfringens strains isolated from horses with anterior enteritis are of type D.     

Conventional antibody against Nepsilon-(carboxymethyl)lysine (CML) shows cross-reaction to Nepsilon-(carboxyethyl)lysine (CEL): immunochemical quantification of CML with a specific antibody

Immunological strategies for the detection of N(epsilon)-(carboxymethyl)lysine (CML), one of the major antigenic structures of advanced glycation end products (AGE), are widely applied to demonstrate the contribution of CML to the pathogeneses of diabetic complications and atherosclerosis. Recent studies have indicated that methylglyoxal (MG), which is generated intracellularly through the Embden-Meyerhof and polyol pathways, reacts with proteins to form MG-derived AGE structures such as N(epsilon)-(carboxyethyl)lysine (CEL). In order to accurately measure the CML contents of the proteins by means of an immunochemical method, we prepared CML-specific antibodies since conventionally prepared polyclonal anti-CML antibody and monoclonal anti-CML antibody (6D12) cross-reacted with CEL. To prepare polyclonal CML-specific antibody, CML-keyhole limpet hemocyanin (CML-KLH) were immunized with rabbit and CEL-reactive antibody was removed by CEL-conjugated affinity chromatography. Monoclonal antibody specific for CML (CMS-10) was obtained by immunization with CML-KLH, followed by successive screening according to CML-bovine serum albumin (CML-BSA)-positive but CEL-BSA-negative criteria. Both polyclonal CML-specific antibody and CMS-10 significantly reacted with CML-proteins but not with CEL-proteins. It is likely therefore that these antibodies can recognize the difference of one methyl group between CML and CEL. Moreover, CMS-10 significantly reacted with BSA modified with several aldehydes and its reactivity was highly correlated with the CML content, which was determined by high performance liquid chromatography, whereas 6D12 showed a low correlation. These results indicate that CMS-10 can be used to determine the CML contents of modified proteins in a more specific way.     

In vitro effects of Clostridium perfringens type D epsilon toxin on water and ion transport in ovine and caprine intestine

Clostridium perfringens type D produces enterotoxaemia in sheep, goats and other animals. The disease is caused by C. perfringens epsilon toxin, and while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by sheep than goat intestine. In an attempt to clarify this, we examined the in vitro effects of epsilon toxin on sheep and goat intestine. Pieces of intestinal mucosa from recently slaughtered animals were mounted in a modified Ussing-type chamber where net water flux (J(w)), short-circuit current (I(sc)) and tissue conductance (G(t)) were simultaneously recorded. After 70 min of incubation with epsilon toxin a reduction in absorptive J(w) and an increase in I(sc) and G(t) were observed in colonic tissues of both sheep and goats, but no alterations were registered in the ileum of either species. These in vitro results show that epsilon toxin affects the transport function of the colonic mucosa but it does not seem to produce any transport alteration in the ileum mucosa.     

Structural relationship between DNA polymerases epsilon and epsilon* and their occurrence in eukaryotic cells.

Monoclonal antibodies raised against the N-terminal half of human DNA polymerase epsilon bind both to a large > 200 kDa form of DNA polymerase epsilon from HeLa cells and to a small 140 kDa form (DNA polymerase epsilon*) from calf thymus, while antibody against the C-terminal half binds to DNA polymerase epsilon but does not bind to DNA polymerase epsilon*. These results indicate that the two enzymes have common structural motifs in their N-terminal halves, and that DNA polymerase epsilon* is very likely derived from DNA polymerase epsilon by removal of its C-terminal half. DNA polymerase epsilon as well as DNA polymerase epsilon* was detected in extracts from cells of numerous eukaryotic species from yeast to human. The results indicate that DNA polymerase epsilon and its tendency to occur in a smaller form, DNA polymerase epsilon*, are evolutionarily highly conserved and that DNA polymerase epsilon may occur universally in proliferating eukaryotic cells.     

Detection by polymerase chain reaction of Clostridium perfringens producing epsilon toxin in faeces and in gastrointestinal contents of goats

F.A. UZAL, J.J. PLUMB, L.L. BLACKALL, D. O'BOYLE AND W.R. KELLY. 1996. A polymerase chain reaction (PCR) was used to identify the gene-encoding epsilon toxin production in Clostridium perfringens types B and D in faeces and in gastrointestinal contents of goats. The samples were cultured in thioglycollate broth and centrifuged. The upper layer of the pellet was used as a template for PCR, obviating the need for DNA extraction. This technique specifically differentiated Cl. perfringens types B and D from Cl. perfringens types A and C and from Escherichia coli . When used to identify Cl. perfringens type D in samples artificially spiked with the micro-organism, the PCR detected as few as 1.4 × 10² cfu g⁻¹ of sample. Gastrointestinal contents and faeces were collected from 20 goats at slaughter and processed by PCR. Several positive results were obtained from the first five goats that were slaughtered and sampled a few days after their arrival at the abattoir, but only a few samples gave positive results during the following weeks, after the goats had been fed a concentrated ration containing monensin. A possible role of this drug in control of enterotoxaemia is suggested.     

Clostridium perfringens toxin types from wild-caught Atlantic cod (Gadus morhua L.), determined by PCR and ELISA

Ninety-five fecal samples from Atlantic cod (Gadus morhua L.), caught along the northern Norwegian coast, were examined bacteriologically for occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. In addition, a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of C. perfringens alpha, beta, and epsilon toxin was used. Clostridium perfringens could be isolated in 37 fecal samples (38.9%) from cod. All isolates were C. perfringens toxin type A (alpha toxin positive) as determined by PCR and also ELISA. In addition, in isolates from two cod (2.1%) the gene encoding for beta2 toxin was found (A, beta2) by PCR. Genes encoding for beta, epsilon, and iota toxins and enterotoxin were not found. This is the first detection of C. perfringens alpha and beta2 toxin in cod and of beta2 toxin in fish in general. The origin of this bacterium in cod is discussed.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Monoclonal antibodies to murine CD3 epsilon define distinct epitopes, one of which may interact with CD4 during T cell activation

The TCR is comprised of two variable chains that confer specificity, called alpha:beta or gamma:delta, physically associated with five different molecules that comprise the complex known as CD3. Antibodies to this complex are very useful, as they react with all T lymphocytes. A rat mAb to mouse CD3 has been prepared. It reacts with 100% of T cells in all mouse strains tested but with no other cell type. It binds to the CD3 epsilon chain. This antibody activates cloned T cell lines and normal T cells, provided suitable accessory cells and signals are present. This antibody detects a determinant similar to but not identical with those detected by two previously reported hamster anti-CD3 epsilon antibodies. This antibody fixes C efficiently, and it is thus useful for depletion of T cells from bulk populations. Activation of T cells by one of the three different anti-CD3 epsilon antibodies was inhibited by the Fab fragment of anti-CD4, similar to the effects of anti-CD4 Fab on two previously reported anti-TCR V region antibodies that bind a CD3 epsilon-associated epitope. This further defines a site involving TCR V regions and CD3 epsilon with which CD4 appears to associate during T cell activation.     

Effects of Clostridium perfringens alpha and epsilon toxins in the bovine gut

Clostridium perfringens alpha and epsilon toxins produce enterotoxaemia in sheep and goats. However, the information regarding the pathophysiology of alpha and epsilon toxins in the bovine intestine is still scanty. In this study, intestinal loops were performed in the ileum and colon of three one-week-old Holstein and two four-week-old crossbreed calves. Laparotomy was performed in all calves under anaesthesia and four loops -three cm long- were performed in the small and large intestines. For both intestines, loops were inoculated with alpha or epsilon toxins. Tissue samples from all loops were obtained and processed for routine histology and for transmission electron microscopy. Congestion was observed in toxin treated loops. Fluid accumulation in the gut lumen was prominent in all treated loops, but in epsilon treated ones the mucous was also haemorrhagic. The histology revealed large amount of exfoliated epithelial cells in the lumen of alpha toxin treated loops and severe haemorrhage was observed in the lamina propria of epsilon toxin treated colonic loops. Despite some necrotic exfoliated enterocytes, no ultraestructural changes were observed in alpha toxin treated loops, though with epsilon toxin the loops exhibited dilation of the intercellular space in the mucosa of both, small and large intestines. These observations indicate that both, alpha and epsilon toxins can alter the intestinal barrier, in calves and are pathogenic for this species.     

PCR detection of Clostridium perfringens producing different toxins in faeces of goats

A polymerase chain reaction (PCR) was used to identify the genes encoding the major toxins of Clostridium perfringens in faeces of goats. When pure cultures of Cl. perfringens types A, B, C, D and E were used as templates in the PCR, amplicons were observed on the agarose gel as bands at approximately the 247 (alpha primers), 1025 (beta primers), 403 (epsilon primers) and 298 (iota primers) bp level of the DNA marker. When used to identify different types of Cl. perfringens in samples artificially spiked with these micro-organisms, the PCR detected as few as 1–1·5×10² cfu g⁻¹ of the five types of Cl. perfringens tested. The PCR technique allowed the identification and typing of Cl. perfringens strains in faeces of goats, without recourse to other techniques such as the mouse neutralization test.     

Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes

A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.