Cytotoxicity assay as a potential alternative method for animal testing for batch release of Italian fish autogeneous vaccines.

Background: Vaccination is widely used in fish aquaculture for three primary reasons: to prevent bacterial disease spreading, minimize antibiotic use and fight antibiotic resistance. Vaccine production is an expensive and consuming process, mainly in terms of money, resources, and animals for quality control. The replace, reduce, and refine (3Rs) philosophy suggests developing and validates alternative methods to animal testing for scientific purposes, even for biologicals and vaccines. Aim: The current study explored the potential use of mouse and fish cells in the in vitro toxicity grade assessment through different methods, as an alternative assay to in vivo residual toxicity tests for autogenous fish vaccine control. Methods: BF2 and L929 cell lines were exposed to vaccine dilutions in two different ways of administration and toxicity grade was recorded by MTS assay, compared to the in vivo gold standard test. Results: Autogenous vaccines (AVs) caused no reactions in the in vivo test. In the in vitro assay, the different toxicity grade recorded was statistically significant between the cell lines adopted and the AVs way of administration. Conclusions: Data obtained represent the first application of 3Rs method to fish AVs produced in Italy, more investigations are needed to collect solid results and standardize new in vitro methods for vaccine quality control.

The Stromal Vascular Fraction from Canine Adipose Tissue Contains Mesenchymal Stromal Cell Subpopulations That Show Time-Dependent Adhesion to Cell Culture Plastic Vessels.

Adipose-derived mesenchymal stromal cells (MSCs) are extensively studied in both human and veterinary medicine. Their isolation is usually performed by collagenase digestion followed by filtration and removal of nonadherent tissue remnants 48 h after seeding. We observed that waste tissue fragments contain cells that adhere belatedly to the plastic. We aimed to investigate their basic properties to speculate on the possible existence of MSC subpopulations. Adipose tissue from three dogs was enzymatically digested. Three cell populations that adhered to the culture plastic 48, 96, and 144 h after seeding were obtained. After expansion, they were analyzed by flow cytometry for MSC-positive (CD90, CD44, and CD29) and -negative (CD14, MHCII, and CD45) markers as well as for endothelial, pericyte, and smooth muscle cell markers (CD31, CD146, and alpha-SMA). Furthermore, cells were assessed for viability, doubling time, and trilineage differentiation ability. No significant differences were found between the three subpopulations. As a result, this procedure has proven to be a valuable method for dramatically improving MSCs yield. As a consequence of cell recovery optimization, the amount of tissue harvested could be reduced, and the time required to obtain sufficient cells for clinical applications could be shortened. Further studies are needed to uncover possible different functional properties.

Expression of a recombinant ASFV P30 protein and production of monoclonal antibodies.

Background: African Swine Fever (ASF) is an infectious disease that affects domestic pig and wild boar populations. The ASF Virus (ASFV) has a genome characterized by a very complex DNA (170-193 kb) that encodes for more than 200 different proteins. Among these, the highly immunogenic phosphoprotein p30 plays a fundamental role in the induction of specific antibodies. To date, the lack of a vaccine against the disease requires continuous studies to improve knowledge about the virus and the development of new tests in addition to virological ones. Aim: The aim of this work was to produce specific monoclonal antibodies (mAbs) against the p30 protein of ASFV, which could find useful applications in routine diagnostics and the implementation of new diagnostic tools. Methods: ASFV p30 encoding gene was amplified and used for the generation of the recombinant baculovirus by transfection of the Sf21 insect cells. The recombinant protein was analyzed by immunofluorescence assay, purified, and used for mice Balb-c immunization. The hybridomas obtained were cultured and screened, using an indirect Enzyme-linked Immunosorbent Assay (iELISA), in order to select clones that secrete the mAbs of interest. Results: The expression of recombinant p30 protein was assessed using direct immunofluorescence. The purified p30 protein fractions were analyzed by Coomassie gels staining confirming the presence of bands with a molecular weight of 30 kDa and used for the immunization of Balb-c mice. Six clones of pure hybridomas secreting the specific mAbs against recombinant p30 were obtained and tested in iELISA. The mAbs were also characterized by Western blot and immunofluorescence assay. The best results were obtained with the anti-p30 mAb 2B8E10 clone which showed high reactivity with both recombinant and viral p30 protein, respectively. Conclusion: In this work, a recombinant p30 protein produced in an insect cell system was purified and used to immunize Balb-c mice. Six anti-p30 mAbs-secreting hybridomas clone cells were obtained. These mAbs displayed high reactivity against the recombinant protein, but only 2B8E10 mAb showed excellent functionality against the p30 protein produced by ASFV. These results open the possibility to develop different diagnostic assays.

A new Montanide™ Seppic IMS1313-adjuvanted autogenous vaccine as a useful emergency tool to resolve a Salmonella enterica subsp. enterica serovar abortus equi abortion outbreak in mares.

Background: In Italy, an autogenous registered vaccine, adjuvanted with aluminum hydroxide, can be administrated to contrast Salmonella enterica subsp. enterica serovar abortus equi infection, coupled to a specific antimicrobial treatment. Case Description: Here, we report the case of an abortion outbreak by Salmonella abortus equi in Central Italy where mares were vaccinated but immediately developed a strong local reaction, maybe due to the adjuvant. Promptly, another autogenous vaccine, substituting the aluminum hydroxide with a new generation adjuvant (Montanide™ Seppic IMS1313), was produced and administrated. The new formulated vaccine did not cause any adverse outcome and conferred high protection titers against the infection. To the best of our knowledge, this is the first reported case of immunization by a vaccine adjuvanted with Montanide™ Seppic IMS1313 in horses. Conclusion: This approach may be used as a preventive strategy for further outbreaks in association with the application of recommended biosafety principles.

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.

Spectroscopic Characterization of Bovine, Avian and Johnin Purified Protein Derivative (PPD) with High-Throughput Fourier Transform InfraRed-Based Method.

Tuberculins purified protein derivatives (PPDs) are obtained by precipitation from heat treated mycobacteria. PPDs are used in diagnosis of mycobacterial infections in humans and animals. Bovine PPD (PPDB) is obtained from Mycobacterium bovis (Mycobacterium tuberculosis complex), while Avian PPD (PPDA) and Johnin PPD (PPDJ) are extracted, respectively, from Mycobacterium avium and M. avium subsp. paratuberculosis (M. avium complex). PPDB and PPDA are used for bovine tuberculosis diagnosis, while PPDJ is experimentally used in the immunodiagnosis of paratuberculosis. Although PPDs date back to the 19th Century, limited knowledge about their composition is currently available. The goal of our study was to evaluate Fourier Transform InfraRed (FTIR) spectroscopy as a tool to differentiate PPDB, PPDA, and three PPDJs. The results highlighted that the three PPDs have specific profiles, correlated with phylogenetic characteristics of mycobacteria used for their production. This analysis is eligible as a specific tool for different PPDs batches characterization and for the assessment of their composition. The entire PPD production may be efficiently controlled, since the N content of each preparation is related to IR spectra, with a reference spectrum for each PPD and a standardized analysis protocol.

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.

Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies.

BACKGROUND: Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. RESULTS: In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2Δ1-25-His6) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2Δ1-25-His6 was obtained after purification by Ni2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2Δ1-25-His6. Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. CONCLUSIONS: The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

First assessment of classical swine fever marker vaccine candidate CP7_E2alf for oral immunization of wild boar under field conditions.

Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have been carried out using live attenuated vaccines that do not allow serological differentiation of infected from vaccinated animals (DIVA). Although this drawback is acceptable for wild boar, the use of marker vaccines would facilitate studies on disease and vaccination dynamics. Recently, the CSF marker vaccine candidate CP7_E2alf was assessed for oral immunization under laboratory conditions. Promising results prompted efforts to study the vaccine candidate under field conditions and in bait formulation. In this context, two oral vaccination campaigns were carried out with CP7_E2alf bait vaccines in two areas called 'faunistic-hunting farms' in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. As a comparator, seven wild boar were hand-fed with baits under laboratory conditions. In the field, bait uptake ranged from 63.7% to 98.7%, whereas antibody prevalence reached only 33.3-35.1%. The marker serology showed a strong influence of sample quality on the test outcome with a total of 85% of samples being classified correctly. Vaccine virus was not detectable. Under hand feeding conditions, six out of seven wild boar took up at least one bait, and five of them showed detectable antibody levels seven weeks after vaccination. These results were supplemented by stability tests. Appropriate stability of vaccine virus was shown both under field and laboratory conditions. In total, most results were in line with our expectations. However, optimization of the DIVA assay has to be attempted in the future.

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.

Percentage of reactor animals to single comparative cervical intradermal tuberculin (SCCIT) in small ruminants in Punjab Pakistan.

To investigate the percentage reactor animals to SCCIT in sheep and goat the present study was carried out by using bovine and avian PPDs at 7 Livestock Experiment Stations of Punjab and villages around two cities. The overall percentage of reactor animals to SCCIT at farms in sheep and goat was 0.9% and 2.4%, while it was 0.4% and 0.0% around two cities, respectively. Hundred percent of goat and 86% of sheep farms under study had positive reactor animals. The reactor animals to SCCIT around two cities were 0% in goat and 0.4% in sheep. Goats (P<0.05) and sheep (P<0.001) kept with large ruminants at farms had higher percentage of reactor animals. Age showed significant (P<0.01) association with tuberculosis in sheep and goat. In goats of more than 6 years of age, the reactor animals to SCCIT were 5, 8 and 11 times higher than goats of 4.1-6, 3-4 and <3 years of age. The reactor animals were significantly (P<0.05) higher in goats of <30kg (3.5%) and>40kg (3.5%) body weights, while these were less in goats weighing between 30 and 40kg (1.5%). Sheep and goat breeds showed significant (P<0.001; P<0.05) difference in percentage of reactor animals to SCCIT. Beetal (3.9%) and Teddy breeds (2.9%) had 3.3 and 2.4 times higher percentages of reactor animals to SCCIT as compared to Pak-Angora (1.2%) breed. However, no animal of Nachhi and Dera Din Pinah breeds showed positive reaction to PPDs.

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.