Identification and Characterization of Clostridium perfringens Atypical CPB2 Toxin in Cell Cultures and Field Samples Using Monoclonal Antibodies.

A direct sandwich enzyme-linked immunosorbent assay (sELISA) was developed for the detection of the atypical ß2-toxin (CPB2) of Clostridium perfringens. Polyclonal (PAbs) and monoclonal (MAbs) antibodies were previously obtained employing recombinant CPB2 produced in the baculovirus system as antigen. In the current study, PAbs were used as capture molecules, while purified MAbs conjugated to horseradish peroxidase (MAbs-HRP) were used for the detection of atypical CPB2 toxin. MAbs 5C11E6 and 2G3G6 showed high reactivity, sensitivity and specificity when tested on 232 C. perfringens cell culture isolates. In addition, a reactivity variation among different strains producing atypical CPB2 toxin was observed using the conformation-dependent MAb 23E6E6, suggesting the hypothesis of high instability and/or the existence of different three-dimensional structures of this toxin. Results obtained by sELISA and Western blotting performed on experimentally CPB2-contaminated feces revealed a time-dependent proteolytic degradation as previously observed with the consensus allelic form of CPB2. Finally, the sELISA and an end-point PCR, specific for the atypical cpb2 gene, were used to test field samples (feces, rectal swabs and intestinal contents) from different dead animal species with suspected or confirmed clostridiosis. The comparison of sELISA data with those obtained with end-point PCR suggests this method as a promising tool for the detection of atypical CPB2 toxin.

Molecular Characterization of Clostridium perfringens Strains Isolated in Italy.

Clostridium (C.) perfringens is the causative agent of several diseases and enteric infections in animals and humans. The pathogenicity of the bacterium is largely mediated by the production of a wide range of toxins. Individual C. perfringens strains produce only subsets of this toxin repertoire, which permits the classification in seven toxinotypes (A-G). In addition, a variety of minor toxins further characterizes the single strains. The aim of this work was to evaluate, using Polymerase Chain Reaction (PCR) assays, the diversity of 632 C. perfringens strains isolated in Italy over 15 years. The genotyped strains were analyzed to determine the presence of major and minor toxins (cpe, consensus, and atypical cpb2), their geographical origins, and the source of isolation (animal species or food). Our study shows that toxinotype A had the greatest representation (93%) and correlated mainly with consensus cpb2 in a variety of animal species, as well as with atypical cpb2 in the five food samples. Type D, associated with cpe and atypical cpb2 minor toxins, was identified in 3% of the cases, and type F was identified in 2.5%. Seven type C isolates (1.1%) were detected in cattle, whereas the only type B atypical cpb2 isolated in Italy was detected in a goat, and one type E cpe+atypical cpb2 was detected in a sheep. Type G was not detected.

Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies.

BACKGROUND: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms. RESULTS: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC. CONCLUSIONS: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze-drying rabbit (Oryctolagus cuniculus) spermatozoa.

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

Role of conserved cysteine residues in the CAIC motif of the SU glycoprotein in the maturation and fusion activity of bovine leukaemia virus.

The surface (SU) and transmembrane (TM) glycoproteins of many retroviruses are linked by disulphide bonds, and the interaction of SU with a cellular receptor results in disulphide bond isomerisation triggered by the CXXC motif in SU. This reaction leads to the fusion of viral and host cell membranes. In this work, we show that the cysteine at amino acid position 212 in the CAIC motif of the SU glycoprotein of bovine leukaemia virus has a free thiol group. A C-to-A mutation at position 212, either individually or in combination with a C-to-A mutation at position 215, was found to inhibit the maturation process, suggesting its involvement in the formation of the covalent bond with TM.

First evaluation of endotoxins in veterinary autogenous vaccines produced in Italy by LAL assay.

Endotoxin contamination is a serious concern for manufacturers of biological products and vaccines in terms of not only quality but also safety parameters. We evaluated the endotoxin presence in different veterinary autogenous vaccines produced by the Pharmaceutical Unit at the Experimental Zooprophylactic Institute of Umbria and Marche "Togo Rosati" (IZSUM). According to the 3Rs principles (Replace, Reduce, Refine), which aim to progressively reduce animal use in the quality control process, we tested the vaccines obtained from gram-negative bacteria and adjuvants by the limulus amebocyte lysate (LAL) assay. The results revealed low endotoxin concentrations compared to available data in the literature and represent the first report of the application of the 3Rs principles to veterinary autogenous vaccines production in Italy.

Evaluation of immune responses in mice and sheep inoculated with a live attenuated Brucella melitensis REV1 vaccine produced in bioreactor.

The Brucella melitensis REV1 vaccine is the most widely employed vaccine for prophylaxis against brucellosis in sheep and goats. The objective of vaccination is disease control in herds or preventing infection in farms. In this study, we produced REV1 vaccine with a protocol, based on the use of liquid medium in a bioreactor, that resulted efficient, safe, relatively fast, and cost-effective. The live attenuated vaccine produced was tested in mice and sheep to investigate its immunogenicity and efficacy. Seventy-two female BALB/c mice were obtained and subdivided in 2 groups, one was stimulated with 1 × 106 colony-forming units (CFUs) of B. melitensis while the other with physiological solution alone and acting as control group. Furthermore, 25 sheep were subdivided into 5 groups: four were inoculated with a B. melitensis dose, ranging from 0.6 × 109 and 3.2 × 109 CFUs and the other was the control group. In addition, a serological diagnosis was performed for sheep by rapid serum agglutination and the complement-fixation test. Immunocompetent cells from both experiment were collected at different times post vaccination and immunostained to evaluate innate and adaptive-immune responses. In mice flow cytometry was used to detect macrophages, T lymphocytes, dendritic cells, memory cells, naïve cells, natural killer cells, major histocompatibility complex type II, B lymphocytes, regulatory T lymphocytes, T helper lymphocytes, cytotoxic T lymphocytes and recently activated CD4+ and CD8+ lymphocytes. In sheep, macrophages, T helper cells, cytotoxic T lymphocytes, regulatory T lymphocytes, dendritic cells, memory cells and naïve lymphocytes, by the same method, were analyzed. The results showed, both in mice and sheep, that the live, attenuated REV1 vaccine stimulated all immunocompetent cells tested, with a balanced innate and adaptive response. In the sheep experiment, the administered vaccine dose was very important because, at the lower doses, immunological tolerance tended to disappear, while, at the highest dose, the immunological tolerance remained active for a long period. In our experimental conditions, the optimal vaccine dose for sheep was 3.2 × 109 CFUs, although a good immune response was found using a dose of 1.6 × 109 CFUs. The vaccine produced in this study could be extensively employed in developing countries to control the brucellosis in sheep and goats.

Single N-glycosylation site of bovine leukemia virus SU is involved in conformation and viral escape.

The bovine leukaemia virus (BLV) envelope protein (Env) is synthesized as a polyprotein precursor (gp72) proteolytically cleaved into the mature surface (SU) and transmembrane (TM) glycoproteins. The amino-terminal region of SU contains conformational epitopes F, G and H, which require a glycosylated SU to be recognized by monoclonal antibodies (MAbs) and antibodies from BLV-infected cattle. The SU contains eight asparagine (N) residues that are putative N-glycosylation sites. The N129, N203, N230 and N251 appear involved in carbohydrate binding, play an essential role in the in vitro infection. To determine which sites were actually glycosylated, we generated mutated SU forms, where each N-glycosylation site was changed to alanine (A). Subsequently, these N to A mutations were inserted into the env gene to generate Env mutants. The increase of electrophoretic mobility of EnvA256 and EnvA271 derived SU showed that the asparagine residues N256 and N271 were also glycosylated. ELISA revealed that only the N129 oligosaccharide determined the antigenic conformation of SU. The syncytium formation induced by EnvA129 showed that fusogenic capacity was independent of amino-terminal SU glycan conformational structure. Finally, anti-BLV serum inhibited syncytia formation even with the EnvA129 mutant. The latter inhibition was higher than Env, suggesting that the oligosaccharides could be also involved in the glycan shield for viral escape.

Identification of a novel overlapping sequential E epitope (E') on the bovine leukaemia virus SU glycoprotein and analysis of immunological data.

Bovine leukaemia virus (BLV), an oncogenic C-type retrovirus, is the causative agent of enzootic bovine leucosis. Binding of BLV to its cellular receptor is mediated by the surface envelope glycoprotein subunit (SU). Previous studies have identified eight different epitopes (A through H) on the BLV SU. In this study, a new sequential epitope was identified using the monoclonal antibody 2G7 (MAb 2G7) on the C-terminal region of the BLV SU. To localise and refine the map of this epitope, a series of deleted forms in the C and N-terminal ends of the glycoprotein were made and synthesised in baculovirus and Escherichia coli expression systems. The synthetic proteins were analysed both in Western blot and MAb-capture ELISA assays. MAb 2G7 recognised a stretch of 11 amino acids, named epitope E', corresponding to residues 189-SDWVPSVRSWA-199 (comprising the 33 amino acids signal peptide) overlapping with the E epitope of the SU. The data obtained by Enzyme-Linked Immunosorbent Assay (ELISA) revealed that the E' epitope was hidden on whole BLV particles and that the variation in reactivity between epitope E' and MAb 2G7 depends on the glycosylation state of SU. Similarly, the analysis of immunological data evidenced that the failure of interaction between the MAb anti-DD' and its epitope was also due to a steric hindrance of the glycosylation. Finally, the ELISA assay analysis performed with the deleted and mutated forms of rSU evidenced that the conformational epitopes F, G and H lied into in the 34-173 amino-acids residues of N-terminal region of SU.

CA 15-3 cell lines and tissue expression in canine mammary cancer and the correlation between serum levels and tumour histological grade.

BACKGROUND: Mammary tumours are the most common malignancy diagnosed in female dogs and a significant cause of mortality and morbidity in this species. Carbohydrate antigen (CA) 15-3 is a mucinous glycoprotein aberrantly over-expressed in human mammary neoplasms and one of the most widely used serum tumour markers in women with breast cancer. The aim of this study was to investigate the antigenic analogies of human and canine CA 15-3 and to assess its expression in canine mammary cancer tissues and cell lines. Immunohistochemical expression of CA 15-3 was evaluated in 7 canine mammary cancer cell lines and 50 malignant mammary tumours. As a positive control, the human breast carcinoma cell line MCF7 and tissue were used. To assess CA 15-3 staining, a semi-quantitative method was applied. To confirm the specificity and cross-reactivity of an anti-human CA 15-3 antibody to canine tissues, an immunoblot analysis was performed. We also investigated serum CA 15-3 activity to establish whether its expression could be assigned to several tumour characteristics to evaluate its potential use as a serum tumour marker in the canine mammary oncology field. RESULTS: Immunocytochemical analysis revealed CA 15-3 expression in all examined canine mammary cancer cell lines, whereas its expression was confirmed by immunoblot only in the most invasive cells (CMT-W1, CMT-W1M, CMT-W2 and CMT-W2M). In the tissue, an immunohistochemical staining pattern was observed in 34 (68%) of the malignant tumours. A high statistical correlation (p = 0.0019) between serum CA 15-3 levels and the degree of tumour proliferation and differentiation was shown, which indicates that the values of this serum marker increase as the tumour stage progresses. CONCLUSIONS: The results of this study reveal that CA 15-3 is expressed in both canine mammary tumour cell lines and tissues and that serum levels significantly correlate with the histological grade of the malignancy.

Purification by Strep-Tactin affinity chromatography of a delete envelope gp51 protein of Bovine Leukaemia virus expressed in Sf21 insect cells.

Bovine leukaemia virus (BLV) causes disease in cattle and it is related to human T lymphotrofic viruses HTLV-1 and HTLV-2. The objective of this study was to express and purify deleted and stable forms of the gp51 envelope glycoprotein of BLV using a baculovirus system. Two forms of the gp51 were synthesised: one comprised the gp51 N-terminal 174 amino acids and a single 6xHis tag (Delta(175-268)gp51-His) and the second form contained the same amino acid sequence and a C-terminal Strep-tag II in addition to the 6xHis tag (Delta(175-268)gp51-STH). The two proteins were expressed and purified by immobilized metal-affinity chromatography (IMAC) or by Strep-Tactin column. The Strep-Tactin technology was more efficient than IMAC method and achieved a high pure recombinant deleted gp51. In addition the Delta(175-268)gp51-STH protein was further concentrated by IMAC. This purified antigen could be used for the isolation of immunoreactive molecules and to develop a competitive ELISA test.

Abortion due to Salmonella enterica serovar Abortusovis (S. Abortusovis) in ewes is associated to a lack of production of IFN-gamma and can be prevented by immunization with inactivated S. Abortusovis vaccine.

Salmonellosis due to Salmonella enterica serovar Abortusovis (S. Abortusovis) is mainly characterized by abortion in sheep. Little is known about the immune response, which develops in the host as a result of infection. We evaluated the immune response of pregnant ewes vaccinated and successively exposed to full virulent S. Abortusovis. We found that vaccine constituted by inactivated S. Abortusovis induced both humoral and cellular-mediated immune response and that it provided protection against a challenge infection due to a fully virulent S. Abortusovis. Furthermore, we found an association between the lack of capability to produce IFN-gamma and abortion. This evidence suggests that protection against abortion can be associated to an IFN-gamma mediated mechanism. Our findings represent an interesting insight to better understand the interplay between host and S. Abortusovis and the effector mechanisms underpinning immune-based protection.

A structural model of 20S immunoproteasomes: effect of LMP2 codon 60 polymorphism on expression, activity, intracellular localisation and insight into the regulatory mechanisms.

The immunoproteasome subunit low molecular weight protein 2 (LMP2) codon 60 polymorphism has been associated with autoimmune diseases. It has also been demonstrated to influence susceptibility to TNF-alpha-induced apoptosis in blood cells and proteasome activity in aged human brain. In the present study, an in silico model of immunoproteasome was used to examine the effect of the R60H polymorphism in the LMP2 subunit. The investigation of immunoproteasome expression, activity and intracellular localisation in an in vitro cellular model, namely lymphoblastoid cell lines, showed no major variations in functionality and amount, while a significant difference in antibody affinity was apparent. These data were integrated with previous results obtained in different tissues and combined with a structural model of the LMP2 polymorphism. Accordingly, we identified three prospective mechanisms that could explain the biological data for the polymorphism, such as modulation of the binding affinity of a putative non-catalytic modifier site on the external surface of the immunoproteasome core, or the modification of any channel between alpha and beta rings.

Assays of proteasome activity in relation to aging.

Proteasomes play a major role in intracellular protein turnover. They exist in cells in several different molecular forms including 20S proteasomes, 26S proteasomes and PA28-20S proteasome complexes. In this study we have compared the properties of these purified proteasome complexes to try to design assays that will distinguish between the different complexes (26S proteasome, 20S proteasome, PA28-20S proteasome) in cell extracts. Although the different purified complexes were found to have differences in stability, and in their sensitivity to low concentrations of SDS and salt, the results suggest that it is not straightforward to assay selectively for each type of complex in cell extracts. The relative contribution of different proteasome complexes varies in different cell types and there may be other proteases present which hydrolyse the chosen substrate. Proteasome assays carried out under defined conditions allow comparisons of activity in cell extracts as a function of age, but separation by gel filtration on a Superose 6 column was found to be a useful method for determining the level of different proteasome related complexes.