Simone Simionatto

Efficient site-directed mutagenesis using an overlap extension-PCR method for expressing Mycoplasma hyopneumoniae genes in Escherichia coli.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and results in significant economic losses in swine production worldwide. Vaccination is considered to be the most cost-effective strategy for control and prevention of this disease. However, the development of new recombinant subunit vaccines is often hampered by the unusual codon usage of this bacterium. To express M. hyopneumoniae proteins in heterologous systems such as Escherichia coli, the TGA codons that encode tryptophan in M. hyopneumoniae genes need to be replaced with the TGG codon. In this study we employed a modified overlap extension-PCR method for site-directed mutagenesis of selected TGA codons. Primers carrying the appropriate TGA to TGG mutation were employed in a two-step PCR amplification. The mutated PCR products were subsequently cloned into E. coli expression vectors. Using this method, we obtained 14 M. hyopneumoniae genes with up to three TGA to TGG substitutions per gene. Expression of the 10 mutated genes in E. coli was achieved. The method was rapid, simple and highly efficient in introducing the desired mutations in the A+T rich M. hyopneumoniae genes. In conclusion, this modified overlap extension-PCR method is suitable for large-scale site-directed mutagenesis of M. hyopneumoniae genes for heterologous expression.

Cloning and purification of recombinant proteins of Mycoplasma hyopneumoniae expressed in Escherichia coli.

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Vaccination is the most cost-effective strategy for controlling and prevention of this disease. However, investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized species-specific M. hyopneumoniae antigens. In this work, 54 predicted genes encoding proteins with potential to be used as subunit vaccine or antigens in diagnostic tests were selected, amplified by PCR and cloned into Escherichia coli expression vectors. Recombinant protein expression, solubility and yields were analyzed. The majority of the recombinant proteins were expressed in inclusion bodies. After solubilization with urea or N-lauroyl sarcosine, recombinant proteins were purified by Ni(2+) affinity chromatography. This approach allowed purification of thirty recombinant M. hyopneumoniae proteins which will be evaluated as vaccine candidates and/or as antigens to be used in diagnostic tests.

Immunisation of mice with Mycoplasma hyopneumoniae antigens P37, P42, P46 and P95 delivered as recombinant subunit or DNA vaccines

Porcine enzootic pneumonia (PEP), which is caused by the fastidious bacterium Mycoplasma hyopneumoniae, is one of the most economically important diseases in the pig industry worldwide. Commercial bacterins provide only partial protection; therefore, the development of more efficient vaccines against PEP is necessary. In this study, the cellular and humoral immune responses elicited by DNA and recombinant subunit vaccines based on the P37, P42, P46 and P95 antigens of M. hyopneumoniae were evaluated after the intramuscular inoculation of BALB/c mice. The expression of the cytokines INFγ, TNFα and IL1 was evaluated by real-time RT-PCR in splenocytes from vaccinated mice. All antigens delivered as subunit vaccines, especially P42 and P95, and the pcDNA3/P46 DNA vaccine were able to elicit strong immune responses. These vaccines induced cellular immune responses and the production of antibodies able to react with native M. hyopneumoniae proteins. Because both cellular and humoral immune responses were induced, P42 and P95 are promising candidates for a recombinant subunit vaccine and P46 is a promising candidate for a DNA vaccine against PEP.

Development of an indirect ELISA-NcSRS2 for detection of Neospora caninum antibodies in cattle

Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assays negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.

Production and characterization of recombinant transmembrane proteins from Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a chronic respiratory disease which causes significant economic losses to the swine industry worldwide. More efficient strategies for controlling this disease are necessary. In this study, we cloned17 genes coding for transmembrane proteins from M. hyopneumoniae, among which six were successfully expressed in Escherichia coli and had their immunogenic and antigenic properties evaluated. All proteins were immunogenic in mice and sera from naturally infected pigs reacted with the recombinant proteins, suggesting that they are expressed during infection. These antigens may contribute for the development of new recombinant vaccines and diagnostic tests against EP.

Recombinant Secreted Antigens from Mycoplasma hyopneumoniae Delivered as a Cocktail Vaccine Enhance the Immune Response of Mice

ABSTRACT Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), which is a respiratory disease responsible for huge economic losses in the pig industry worldwide. The commercially available vaccines provide only partial protection and are expensive. Thus, the development of alternatives for the prophylaxis of EP is critical for improving pig health. The use of multiple antigens in the same immunization may represent a promising alternative. In the present study, seven secreted proteins of M. hyopneumoniae were cloned, expressed in Escherichia coli, and evaluated for antigenicity using serum from naturally and experimentally infected pigs. In addition, the immunogenicity of the seven recombinant proteins delivered individually or in protein cocktail vaccines was evaluated in mice. In Western blot assays and enzyme-linked immunosorbent assays, most of the recombinant proteins evaluated were recognized by convalescent-phase serum from the animals, indicating that they are expressed during the infectious process. The recombinant proteins were also immunogenic, and most induced a mixed IgG1/IgG2a humoral immune response. The use of these proteins in a cocktail vaccine formulation enhanced the immune response compared to their use as antigens delivered individually, providing evidence of the efficacy of the multiple-antigen administration strategy for the induction of an immune response against M. hyopneumoniae.

Leptospirosis diagnosis using Nested-PCR

Leptospirosis is a worldwide sanitary problem. Its clinical signs resemble that of other diseases like Dengue and Flu, and it is difficult to distinguish between them. Currently available diagnostic methods shown low sensitivity and specificity. Efforts have been made to develop simpler, faster and more efficient diagnostic methods. The aim of this work was to evaluate and optimize a Nested-PCR method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the LipL32 gene. The sensitivity and specificity of the assay was evaluated using seven saprophytic serovars and 35 pathogenic serovars. This technique showed to be very specific for pathogenic serovars, however it lacked sensitivity. In order to enhance the sensitivity, another primer pair was designed which amplify a 183 bp region within the 264 bp region in lipL32 gene, and used in a Nested-PCR assay. This approach was much more sensitive than traditional PCR.

In silico identification of Corynebacterium pseudotuberculosis antigenic targets and application in immunodiagnosis

Caseous lymphadenitis (CLA) is a disease caused by Corynebacterium pseudotuberculosis. It affects mainly small ruminants and causes significant economic losses worldwide. Because symptoms are not immediately noticeable, CLA clinical diagnosis is not effective. Numerous serological tests are being developed to detect the disease in asymptomatic animals, but currently available immunoassays have problems with sensitivity. Current ELISA formats use native bacterial antigens, and recombinant proteins could be useful for improving the immunoassay parameters. The C. pseudotuberculosis proteins CP0126a, CP0369 and CP1957 were identified from 2097 candidate proteins by mature epitope density (MED) analysis, expressed in Escherichia coli and evaluated in an indirect immunoenzymic system. The CP0126a, CP0369 and CP1957 ELISAs showed 77.5 %, 92.5 % and 92.5 % specificity and 95 %, 90 % and 85 % sensitivity, respectively. Receiver operating characteristic (ROC) curve analysis showed an area under the curve of 0.874, 0.951 and 0.881, respectively. The proteins identified in silico were recognized by antibodies in the sera from infected animals without being recognized in negative samples. The ELISA assay using the rCP0369 protein as antigen had the greatest specificity and sensitivity values, followed by rCP1957. This is an interesting strategy for seroepidemiological investigations in sheep flocks due to its significant specificity and sensitivity.

Effect of the Kozak sequence on seroconversion of mice immunized with a DNA vaccine against swine colibacilosis

The neonatal diarrhea in swine caused by enterotoxigenic Escherichia coli (ETEC) is responsible for high mortality and low growth rate in pigs and it is mainly dependent on the capacity of E. coli to attach to the surface of the small intestine, a property mediated by fimbria. In this study the faeC gene, which codes for the minor fimbrial subunit of E. coli K88ab, was cloned in the eukaryotic expression vector pcDNA3, associated or not to the Kozak sequence. Plasmid DNA of the two versions of the vaccine candidate was inoculated in mice by the intramuscular route, in two doses, at 0 and 21 days. The animals that received the DNA vaccine containing faeC associated to the Kozak sequence presented seroconversion significantly higher (P<0.05) than the one vaccinated with pcDNA3/faeC without the Kozak sequence.