Evaluation of the genetic profiles of Brucella melitensis strain from Turkey using multilocus variable number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) techniques.

Brucellosis is caused by Brucella, and Brucella melitensis is highly prevalent in small ruminants in Turkey. Our aim was to genotype 50 B. melitensis strains isolated from sheep, goat, and cattle abortion samples from different farms in seven geographical regions of Turkey between 2009 and 2017. Forty-six different genotypes were detected in 50 isolates studied according to the MLVA-16. Thirty out of 50 isolate profiles matched profiles from the database exactly, and the remaining 20 were absent. Of these 30 isolates, 93.3% were identical to human isolates previously present in the database. All B. melitensis strains belonged to the eastern Mediterranean group. Genotype 43 was the most common isolate profile, and sequence typing (ST8) was dominant and detected in 39 strains. MLST analysis revealed a novel profile in 11 strains. On comparing the sequences of ST8 and the novel ST, a glucokinase gene variation was detected. In the MLST and MLVA analyses, no distinction was made between B. melitensis biovars. Moreover, there was no significant difference between the strains based on host, region, and year. Consequently, the discrimination power of MLVA was higher than that of MLST in this study. Contrastingly, MLST was useful in distinguishing strains according to geographic origins, as determined by performer studies. Profiles determined by MLVA were the same as those in humans. This raises concerns in regard to One Health and transition between hosts, as it is clear that protecting animal health is very important for human health.

Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection.

BACKGROUND: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

A one-year descriptive epidemiology of zoonotic abortifacient pathogen bacteria in farm animals in Turkey.

This study aimed to investigate the epidemiology of 10 suspicious pathogenic bacteria in 250 stomach contents of aborted calf, lamb, and goat foetuses in 2019. The 155 positive samples obtained from PCR consisted of 53 (58.88 %) bacteria from 90 lamb samples, 10 (43.47 %) bacteria from 23 goat samples, and 92 (67.15 %) bacteria from 137 calf samples. The five most common bacteria associated with abortions were Brucella melitensis, 52 (20.9 %); B. abortus, 13 (5.2 %); Leptospira spp., 34 (13.6 %); Campylobacter fetus, 52 (20.9 %); and Coxiella burnetii, 4 (1.6 %). The highest rate of B. melitensis (65.4 %), B. abortus (69.2 %), Leptospira spp. (67.6 %), and C. fetus (50 %) was detected in the aborted calf samples. The highest individual rate was that of C. fetus (5.2 %). The flock-herd rates of B. melitensis, B. abortus, Leptospira spp., C. fetus, and C. burnetii infections in the 29 farms studied were 34.48 %, 20.69 %, 62.06 %, 82.75 %, and 3.44 %, respectively, with a confidence level and interval of 95 %. The frequency of abortions caused by Leptospira spp. and Campylobacter fetus may be related to increasing in B. melitensis. The rates of aborted calf, lamb, and goat foetuses among the various sampling periods and regions were significantly (P < 0.01) different. In conclusion, precautions should be applied to reduce the spread of these bacterial agents in high-risk areas and to eliminate the risk of harbouring these zoonotic infections in humans. Therefore, these results must be taken into account in the development of control and protection strategies against abortions in animals.

Development of Brucella melitensis Rev.1 ΔOmp19 mutants with DIVA feature and comparison of their efficacy against three commercial vaccines in a mouse model.

Brucella is an intracellular zoonotic pathogen that can affect many hosts. Brucella melitensis Rev.1 is a live attenuated, is one of the most effective vaccine strain against brucellosis. It can be used safely in sheep, goats, and even cattle. Although many studies are available on this topic, there is no effective vaccine strain for sheep and goats that distinguishes the antibody titer produced between the field infections and vaccinations. Outer membrane protein 19 (Omp 19) is both virulent and a protective antigen found on the cell-wall of the Brucella strain. In this study, used the suicide plasmid pJQ200KS, which contained homologous region without Omp19 Open Reading Frame (ORF) that was transferred to B. melitensis Rev.1 and further transformed into spheroplasts along with penicillin, ampicillin, and glycine by electroporation. To obtain a mutant vector from Escherichia coli, we used the heat shock transformation method along with the blue-white colony screening using X-gal media, whereas for the gene transfer in Brucella, we used electroporation. A scanning electron microscope (S.E.M) was used to observe the spheroplast transformation while the mutant vector and deletion mutants were confirmed through PCR and sequence analysis. In the mouse model efficacy trials, three commercial vaccines were found to comply with the OIE standards. Although the deletion mutants 19 and 44/10 had similar efficiency as the commercial vaccines in terms of stimulation power, the ELISA test with Omp19 protein showed the same results as the negative control. The Rev.1 Omp19 deletion mutants obtained in this study contained sufficient residual virulence, and their protective immunity was similar to the commercial vaccines. The study showed that a vaccine prepared using a B. melitensis Rev.1 ΔOmp19 can act as a marker vaccine or differentiate infected from vaccinated animals (DIVA) through the ELISA test that detects the Omp19 protein.

Evaluation of Boron's Adjuvant Activity in Inactive Bacterin Vaccines Using the Mice Model.

Vaccination is the most effective, reliable, and economical way of preventing or reducing the effect of infectious diseases. When preparing inactive vaccines, a range of additives called adjuvants are necessary to enhance the magnitude of the immune response. Boron has a wide range of industrial and medical applications, and its positive effects on distinct functions have been described in plants, humans, and animals. However, no studies exist about the possible adjuvant activities of boron compounds in vaccines. Hence, in this study, the potential adjuvant effect of boric acid was explored and compared with common veterinary adjuvants in a mice model. Staphylococcus aureus (S. aureus) used as vaccine antigen was isolated from dairy cows with bovine mastitis. Vaccines adjuvanted with boric acid, aluminum hydroxide, Montanide ISA 50 and ISA 206, and Montanide + boric acid combinations were prepared. The efficacy of vaccines was evaluated according to local reactions at the injection site, C-reactive protein, total Ig G, total Ig M, and anti-S. aureus antibody levels in mice. Boric acid reduced local inflammatory reactions induced by the Montanide adjuvants. Moreover, mice vaccinated with boric acid-adjuvanted vaccine had higher levels of anti-S. aureus antibody than those in the controls (P < 0.05) and were similar to the levels found in mice sensitized with aluminum hydroxide. Total Ig G and Ig M results were, however, unsuitable for the assessment of adjuvant activity for this study. In conclusion, this study revealed that boric acid has an adjuvant potential in inactive bacterin vaccines, but further target animal studies are needed.

Evaluation of random amplified polymorphic DNA and multiple-locus variable number tandem repeat analyses for Mycoplasma cynos.

Mycoplasma spp. can cause diseases of the respiratory system as well as urogenital infections, infertility, and anemia. The members of this genus have a low G + C content compared to other bacteria. Because primers used in the random amplified polymorphic DNA (RAPD) technique are only 10 bp long and have high GC content, this method can be inadequate for genotyping Mycoplasma spp. isolates. The aim of this study was to develop and evaluate multiple-locus variable number tandem repeat analysis (MLVA) and two-primer RAPD (TP-RAPD) procedures for subtyping Mycoplasma cynos isolates. A total of 55 M. cynos isolates obtained from 162 bronchoalveolar lavage fluid samples from shelter and pet dogs were used in this study. Seventy-four tandem repeat regions were detected in the M. cynos genome, and two of these loci were determined to be suitable and used for development of the MLVA scheme. The results of variable number tandem repeat (VNTR) analysis and TP-RAPD-PCR were compared with RAPD-PCR. The discriminatory power of TP-RAPD-PCR (Hunter-Gaston diversity index [HGDI] = 0.84) was higher than those of RAPD-PCR (HGDI = 0.727), VNTR1 (HGDI = 0.8), and VNTR3 (HGDI = 0.757). We observed that the TP-RAPD-PCR and MLVA methods provide clearer data and are more successful in determining genetic diversity, in contrast to the RAPD-PCR method for this species.

Comparison of five methods for isolation of DNA from Mycoplasma cynos.

Extraction of DNA from Mycoplasma cultured on agar medium is difficult because the plasticity of these microorganisms enables agar penetration. This eventually causes cell loss during harvesting of colonies from the agar surface. Here, we used the GenElute™ gel extraction kit, which is usually used to purify polymerase chain reaction products, for extracting DNA from Mycoplasma. We compared the DNA extraction efficiency of the GenElute™ gel extraction kit from Mycoplasma cynos cultured in agar medium with four other DNA extraction methods. The results were evaluated based on the purity and amount of DNA obtained from one Mycoplasma colony. Eight strains of Mycoplasma cynos isolated from the broncho-alveolar lavage fluid of dogs were used. The GenElute™ gel extraction protocol was the most efficient among all the methods tested in this study as it yielded the highest amount and the purest quality of DNA (199.3±0.744ng/µl) from a single colony. Among the methods tested, the GenElute™ gel extraction method is the most rapid, sensitive, and simple method for DNA extraction from Mycoplasma. This procedure may also prove useful for extracting DNA from other Mycoplasma species.

The effectiveness of anti-R. equi hyperimmune plasma against R. equi challenge in thoroughbred Arabian foals of mares vaccinated with R. equi vaccine.

This study aimed to determine the effectiveness of a pregnant mare immunization of a Rhodococcus equi (R. equi) vaccine candidate containing a water-based nanoparticle mineral oil adjuvanted (Montanide IMS 3012) inactive bacterin and virulence-associated protein A (VapA), as well as the administration of anti-R. equi hyperimmune (HI) plasma against R. equi challenge in the mares' foals. The efficacy of passive immunizations (colostral passive immunity by mare vaccination and artificial passive immunity by HI plasma administration) was evaluated based on clinical signs, complete blood count, blood gas analysis, serological response (ELISA), interleukin-4 (IL-4) and interferon gamma (IFN- γ ), total cell count of the bronchoalveolar lavage fluids (BALF) samples, reisolation rate of R. equi from BALF samples (CFU/mL), lung samples (CFU/gr), and lesion scores of the organs and tissue according to pathological findings after necropsy in the foals. The vaccination of pregnant mares and HI plasma administration in the foals reduced the severity of R. equi pneumonia and lesion scores of the organs and tissue by 3.54-fold compared to the control foals. This study thus indicates that immunization of pregnant mares with R. equi vaccine candidate and administration of HI plasma in mares' foals effectively protect foals against R. equi challenge.

Bacterial penetration of restored cavities using two self-etching bonding systems.

OBJECTIVE: The aim of this study was to investigate the effects of two bonding systems, with and without antibacterial monomers, on marginal bacterial and dye leakage. MATERIALS AND METHODS: Class V cavities were prepared in extracted teeth for a bacterial leakage test, and the teeth were sterilized using a steam autoclave. Four cavities were not restored for the controls, and the other teeth were divided into two groups (n = 16 cavities each): Clearfil Protect Bond group (CPB) and Clearfil SE Bond group (CSE). After application of the bonding agent, the cavities were restored using a composite resin (Clearfil AP-X). The teeth were thermocycled, stored in a broth culture of 1.56 × 108 colony forming units (CFU)/ml of Streptococcus mutans at 37°C for 10 days, and subsequently processed for bacterial staining. Sections from the demineralized teeth were evaluated under a light microscope. In the dye leakage test, the cavities were restored as described in the bacterial penetration test. After thermocycling, the teeth were immersed in 5% basic fuchsin for 24 h, and then divided in half and observed under a stereomicroscope. The data were analyzed using the Kruskal-Wallis and Mann-Whitney U-tests (P = 0.05). RESULTS: The bacterial stain was detected at the cavity wall of five cavities in both bonding systems. Additionally, two cavities in the CSE group, one cavity in the CPB group, and all control cavities showed bacterial staining within the cut dentinal tubules. Dye staining at the axial cavity wall was detected in only three of the teeth for both bonding systems. CONCLUSION: The bonding systems used in this study provided an acceptable marginal seal to prevent bacterial and dye leakage.

Assessment of antibacterial activity of EndoREZ.

OBJECTIVE: The aim of this in vitro study was to evaluate antibacterial activity of a new resin based sealer, EndoREZ in comparison with 5 other sealers: AH 26, Diaket, Sultan, Apexit, and RoekoSeal. STUDY DESIGN: The effect of 6 different sealers on the growth of 3 bacteria (Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa) was measured using the agar diffusion test (ADT) and direct contact test (DCT). For ADT, 200 microL bacterial suspensions were spread on agar plates and freshly mixed sealers were applied to uniform wells punched in the agar. The zones of inhibition of bacterial growth were measured at 24 hours, 48 hours, 7 days, and 10 days. For DCT, 2 sets of sealers were prepared: fresh and 24-hour samples. Fresh samples were used within 20 minutes of mixing time while 24-hour samples were allowed to set in a humid atmosphere at 37 degrees C for 24 hours before testing. Sealers were mixed and placed on the walls of microtiter plate wells and 10-microL bacterial suspensions were allowed to directly contact the sealers for 1 hour. Fresh media were added and 15 microL were transferred from this plate to another plate containing fresh medium (215 microL). Bacterial growth of this last plate was then measured using spectrophotometer every hour over 16 hours. RESULTS: ADT results indicated that EndoREZ, Apexit, and RoekoSeal did not show any antibacterial activity. In DCT results, AH 26 and Sultan were potent bacterial growth inhibitors. CONCLUSION: EndoREZ is not as potent a bacterial growth inhibitor as Sultan and AH 26.