Development and evaluation of a blocking ELISA for serological diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) is an important viral disease characterized by persistent infection in equids worldwide. Most EIA cases are life-long virus carriers with low antibody reactions and without the appearance of clinical symptoms. A serological test with high sensitivity and specificity is required to detect inapparent infection. In this study, a B-cell common epitope-based blocking ELISA (bELISA) was developed using a monoclonal antibody together with the EIAV p26 protein labelled with HRP. The test has been evaluated against the standard and with field serum samples globally. This bELISA test can be completed within 75 min, and the sensitivity is higher than those of either the AGID or one commercial cELISA kit. This bELISA assay was 8-16 times more analytically sensitive than AGID, and 2 to 4 times more analytically sensitive than one cELISA kit by testing three sera from the USA, Argentina, and China, respectively. The 353 serum samples from Argentina were tested, in comparison with AGID, the diagnostic sensitivity and specificity of our bELISA assay were 100% (154/154) and 97.0% (193/199), respectively, and the accuracy of the bELISA test was 98.3%. The bELISA test developed in this study is a rapid, sensitive, specific method for the detection of EIAV infection, and could be a promising candidate for use in the monitoring of the EIA epidemic worldwide. KEY POINTS: • A universal epitope-based blocking enzyme-linked immunosorbent assay (bELISA) was developed for detection of antibodies to EIAV. • The bELISA assay can be used to test EIAV serum samples from different regions of the world including North America, South America, Europe, and Asia. • The bELISA assay was evaluated in three different international labs and showed a better performance than other commercial kits.

Development of a Competitive Enzyme-Linked Immunosorbent Assay Based on Purified Recombinant Viral Protein 7 for Serological Diagnosis of Epizootic Haemorrhagic Disease in Camels.

Epizootic haemorrhagic disease virus (EHDV) is a member of the Orbivirus genus in the Reoviridae family, and it is the etiological agent of an arthropod-transmitted disease that affects domestic and wild ruminants. Due to its significant economic impact, many attempts have been done in order to develop diagnostic immunoassays mainly based on the use of the viral protein 7 (VP7), that is, the immunodominant serogroup-specific antigen. In this work, a recombinant VP7 (recVP7) of EHDV serotype 2 was produced in a baculovirus system, and after purification using ion metal affinity chromatography, we obtained a high yield of recombinant protein characterized by a high degree of purity. We used the purified recVP7 as reagent to develop a competitive enzyme-linked immunoassay (c-ELISA), and we tested the presence of EHDV antibodies in 185 dromedary camel serum samples. The c-ELISA showed good performance parameters in recognising positive sera of naturally EHDV-infected dromedary camels; in particular, our developed test reached 85.7% of sensitivity, 98.1% of specificity, 93% of accuracy, and a high agreement value with results obtained by the commercial ELISA kit (Cohen's kappa value of 0.85) that we adopted as the reference method. This c-ELISA could be a useful screening test to monitor the virus spread in camels that are sentinel animals for endemic areas of disease.

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/µL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

MFAP9: Characterization of an extracellular thermostable antibacterial peptide from marine fungus with biofilm eradication potential.

An extracellular thermostable antibacterial peptide designated as MFAP9 was purified from marine Aspergillus fumigatus BTMF9 by ammonium sulfate precipitation followed by ion exchange chromatography on a DEAE-sepharose column. The molecular weight of MFAP9 was found to be∼3 kDa in SDS-PAGE gel corresponding a single intensity peak in MALDI-TOF. The distinct peak with a retention time of 32.5 min appeared in high performance liquid chromatography (HPLC), further confirming the purity. Isoelectric focusing, two-dimensional gel electrophoresis and peptide mass fingerprinting were performed for the characterization of MFAP9. Functional analysis of purified MFAP9 exhibited strong antibacterial activity against Bacillus circulans (NCIM 2107) with MIC and MBC values of 0.525 µg/mL and 4.2 µg/mL, respectively. The in vitro antibiofilm effect of MFAP9 was analyzed against bacteria which have strong biofilm forming potential. The antibiofilm effect of MFAP9 treatment on Bacillus pumilus was examined using scanning electron microscopy. MFAP9 was found to be active at high temperatures and a wide range of pH (28). In addition, it showed varied sensitivity towards proteolytic enzymes. The peptide was nontoxic to human RBCs at higher concentrations. These results indicate that MFAP9 is an antibacterial peptide, suitable for the development of novel anti-infective agent with strong antibiofilm potential.

Humoral antibody response of 10 horses after vaccination against African horse sickness with an inactivated vaccine containing all 9 serotypes in one injection.

BACKGROUND: African horse sickness (AHS) is a devastating viral disease of equids that was first recorded in 1327. Currently, prevention and control of the disease are based on attenuated vaccines and midge control. It has been shown that attenuated Orbivirus vaccines are not always safe as they may reverse to virulence. OBJECTIVES: In the Emirate of Dubai, a vaccination experiment was carried out with an inactivated AHS vaccine produced at the Central Veterinary Research Laboratory (CVRL), Dubai, UAE to investigate the humoral antibody response of AHS-naïve horses to this vaccine. Our vaccination experiment was performed to establish an AHS vaccine bank in the UAE to protect horses from the disease in case of an outbreak. Therefore, CVRL established an inactivated AHS vaccine containing all nine serotypes which induce high neutralising antibodies. STUDY DESIGN: A total of 10 horses kept in a desert isolation area were subcutaneously and intramuscularly vaccinated with an inactivated vaccine containing all nine AHS serotypes previously isolated from Kenyan horse fatalities. Primary immunisation was followed by two booster immunisations 4 weeks and 6 months apart. After 13 months, an annual booster was administered. METHODS: Blood samples were regularly withdrawn for ELISA and virus neutralisation testing. Additionally, EDTA blood was tested every second day for 14 days post each vaccination for the presence of AHS virus or its RNA. RESULTS: Results show that ELISA and virus neutralising antibodies appeared after the first booster, declined after 4-6 months and therefore three vaccinations and an annual vaccination are necessary to achieve high protective virus neutralising antibodies. MAIN LIMITATIONS: No challenge infection was carried out due to the lack of a safe facility in the UAE. CONCLUSION: Before more advanced AHS vaccines become a reality, inactivated vaccines containing all nine serotypes should be used as they produce high ELISA and neutralising antibodies.

Immune response of horses to inactivated African horse sickness vaccines.

BACKGROUND: African horse sickness (AHS) is a serious viral disease of equids resulting in the deaths of many equids in sub-Saharan Africa that has been recognized for centuries. This has significant economic impact on the horse industry, despite the good husbandry practices. Currently, prevention and control of the disease is based on administration of live attenuated vaccines and control of the arthropod vectors. RESULTS: A total of 29 horses in 2 groups, were vaccinated. Eighteen horses in Group 1 were further divided into 9 subgroups of 2 horses each, were individually immunised with one of 1 to 9 AHS serotypes, respectively. The eleven horses of Group 2 were immunised with all 9 serotypes simultaneously with 2 different vaccinations containing 5 serotypes (1, 4, 7-9) and 4 serotypes (2, 3, 5, 6) respectively. The duration of this study was 12 months. Blood samples were periodically withdrawn for serum antibody tests using ELISA and VNT and for 2 weeks after each vaccination for PCR and virus isolation. After the booster vaccination, these 27 horses seroconverted, however 2 horses responded poorly as measured by ELISA. In Group 1 ELISA and VN antibodies declined between 5 to 7 months post vaccination (pv). Twelve months later, the antibody levels in most of the horses decreased to the seronegative range until the annual booster where all horses again seroconverted strongly. In Group 2, ELISA antibodies were positive after the first booster and VN antibodies started to appear for some serotypes after primary vaccination. After booster vaccination, VN antibodies increased in a different pattern for each serotype. Antibodies remained high for 12 months and increased strongly after the annual booster in 78% of the horses. PCR and virus isolation results remained negative. CONCLUSIONS: Horses vaccinated with single serotypes need a booster after 6 months and simultaneously immunised horses after 12 months. Due to the non-availability of a facility in the UAE, no challenge infection could be carried out.

Evaluation of Different Local Drug Delivery Systems in the Management of Chronic Periodontitis: A Comparative Study.

AIM: The present study aimed to assess the use of various local drug delivery systems in the management of chronic periodontitis. MATERIALS AND METHODS: A total of 60 patients aged around 30-55 years were included. The subjects who were enrolled under took a phase I therapy that included scaling and root planing (SRP). Patients who satisfied the conditions for selection to enter the trial were assigned randomly to three groups, with each group consisting of 20 participants as follows: group I: controlled-release drugs-chlorhexidine gel, group II: metronidazole gel, group III: tetracycline fibers. The plaque index (PI), gingival index (GI), and periodontal pocket depth (PPD) were recorded after 1st week as the baseline data and were recorded again after 15 days and 30 days post-baseline. RESULTS: The mean GI scores were 1.32 ± 0.10, 0.88 ± 0.16, and 0.76 ± 0.12, at baseline, 15 days, and 30 days, respectively, in group I. In group II, the mean GI score reduced to 1.09 ± 0.83 at 30 days from 1.48 ± 0.27 at baseline. Likewise, in group III the mean GI score reduced to 0.90 ± 0.62 at 30 days from 1.38 ± 0.06 at baseline. All the groups demonstrated a statistically significant difference at various intervals. The mean PI score decreased to 0.90 ± 0.78 at 15 days from 1.46 ± 0.22 at baseline in group III. A statistically significant difference at different intervals was seen in group III only. In all groups, the intergroup comparison of PPD was found to be statistically significant. CONCLUSION: This study demonstrated that although thorough SRP is an effective treatment method for elimination of chronic periodontal pockets, improved results can be obtained by adjunctive use of locally administered chlorhexidine gel, metronidazole gel, and tetracycline fibers. CLINICAL SIGNIFICANCE: The use of the adjunctive local drug delivery system along with mechanical cleansing in the treatment of periodontal pockets in chronic periodontitis is therapeutically beneficial.

African Horse Sickness Fever in Vaccinated Horses: Short Communication.

Our investigation has shown that multiple vaccinations with inactivated African horse sickness (AHS) vaccines containing all 9 serotypes and produced at the Central Veterinary Research Laboratory in Dubai, UAE, protect horses from AHS. However, the immunization did not prevent African horse sickness fever (AHSF) in approximately 10% of the vaccinated horses despite high enzyme-linked immunosorbent assay and virus neutralizing antibodies. African horse sickness fever is a very mild form of AHS with similar clinical signs. From all 6 horses which had developed AHSF, no virus was isolated from EDTA blood withdrawn during the acute phase of infection. Despite high neutralizing antibodies, serotype 9 was detected by polymerase chain reaction in 4 of them. All 6 horses recovered within 72 hours, after they developed mild clinical signs of AHS.

Evaluation of serological responses in horses challenged with Burkholderia pseudomallei using current diagnostic tests for glanders.

Six horses were challenged experimentally with a strain of Burkholderia pseudomallei isolated from a fatal case of the infection in a dromedary camel years earlier in the Emirate of Dubai. Three horses were inoculated subcutaneously and in 3 the bacterium was administered by the oral route. Four of the horses became serologically positive based on reactions to one or more of the OIE described tests for glanders. B. pseudomallei was re-isolated from the 4 serological positive horses. Only one of the subcutaneously infected horses, developed fever for 3 days. The white blood cell values and the neutrophil counts were also elevated. The study confirmed that existing serological test for diagnosing glanders cannot differentiate between glanders and melioidosis in horses.

Evaluation of Therapeutic Efficacy of Different Treatment Modalities in Oral Submucous Fibrosis: A Comparative Study.

AIM: Aim of this study was to assess the efficiency of different treatment modalities for oral submucous fibrosis. MATERIALS AND METHODS: Sixty patients were included in the study, which was diagnosed as stage II oral submucous fibrosis (OSMF) based on habitual history and clinical findings. Three groups were made after randomization, i.e., group 1: capsule lycopene group, group 2: capsule lycopene and injection dexamethasone, group 3: injection dexamethasone and hyaluronidase group. Symptom severity was done by visual analog scale (VAS) scoring system viz burning sensation/pain in the patients; patient satisfaction was assessed. Vernier calipers were used to measure patients' maximum mouth opening at day 1, 1st month, 2nd month, 3rd month. RESULTS: Male and female had the mean age of 28.20 ± 4.26 and 39.34 ± 2.12 in group 1, in group 2 was 27.88 ± 7.12 and 40.92 ± 7.16, in group 3 was 28.90 ± 8.69 and 40.10 ± 6.22, respectively. There was no statistically significant difference between treatment modalities based on satisfaction. On 2nd month, maximum patients with no pain were more in group 3 followed by group 2, and this was statistically significant. At a 3rd month, the maximum reduction in pain was in group 3 followed by group 2 and group 1. Mouth opening was improved in the group 3 followed by groups 2 and 1, respectively. On 3rd month statistically significant difference was observed between the study groups. CONCLUSION: The present study concludes that the treatment with dexamethasone + hyaluronidase group showed better results in improvement in mouth opening in OSMF patients than lycopene, lycopene and dexamethasone groups. Improvement in mouth opening, reduced burning sensation in OSMF patients was also shown by lycopene. Hence lycopene can be considered as a good alternative for treatment for OSMF when dexamethasone is contraindicated due to different reasons. CLINICAL SIGNIFICANCE: Any oral cavity part can be affected by OSMF including the pharynx. It is a potentially malignant disorder. So early recognition and initiation of the effective regimen for the treatment in both early and advanced cases of OSMF are necessary.

Effectiveness of Mentha piperita Leaf Extracts against Oral Pathogens: An in vitro Study.

AIM: The study aims to assess the Mentha piperita leaf extract's effectiveness against oral pathogens. MATERIALS AND METHODS: The leaf extract of M. piperita was prepared using cold water method. The three microbial strains, i.e., Streptococcus mutans, Aggregatibacter actinomycetem-comitans, and Candida albicans were used as microbiological materials. Chlorhexidine 0.2% was used as positive control. The digital caliper was used to measure the zone of inhibition to know the antimicrobial activity at 24 and 48 hours. To compare the activity within and between the different microbial strains, one-way analysis of variance (ANOVA) was used. To analyze the data, Statistical Package for the Social Sciences (SPSS) software version of 21.0 was used. The p-value ≤0.05 was considered as statistically significant. RESULTS: Maximum inhibition zone was seen in both M. piperita extracts and 0.2% chlorhexidine with S. mutans at 24 and 48 hours, followed by A. actinomycetemcomitans, and C. albi-cans respectively. The statistical analysis ANOVA reveals the statistically significant association of M. piperita extracts with p-value <0.001. The comparison with 0.2% chlorhexidine at 24 hours showed a p-value of <0.04 and at 48 hours, it showed a p-value <0.001, which was statistically significant. CONCLUSION: The present study concluded that M. piperita showed antimicrobial activity against the oral microorganisms which are causing major less or more severe oral diseases and it can be administered as an alternative medicine for the conventional treatment. CLINICAL SIGNIFICANCE: The study results serve as a guide in selecting and providing information about the efficacy of M. piperita extracts to the dental professionals. The discovery of a potential herbal medication would be a great development in the field of antimicrobial therapies.

Serodiagnosis of aspergillosis in falcons (Falco spp.) by an Afmp1p-based enzyme-linked immunosorbent assay.

Aspergillosis in falcons may be associated with high mortality and difficulties in clinical and laboratory diagnosis. We previously cloned an immunogenic protein, Afmp1p, in Aspergillus fumigatus and showed that anti-Afmp1p antibodies were present in human patients with A. fumigatus infections. In this study, we hypothesise that a similar Afmp1p-based enzyme-linked immunosorbent assay (ELISA) could be applied to serodiagnose falcon aspergillosis. A specific polyclonal antibody was first generated to detect falcon serum IgY. Horseradish peroxidase-conjugate of this antibody was then used to measure anti-Afmp1p antibodies in sera collected from falcons experimentally infected with A. fumigatus, and the performance of the Afmp1p-based ELISA was evaluated using sera from healthy falcons and falcons with documented A. fumigatus infections. All four experimentally infected falcons developed culture- and histology-proven invasive aspergillosis. Anti-Afmp1p antibodies were detected in their sera. For the Afmp1p-based ELISA, the mean ± SD OD450 nm using sera from 129 healthy falcons was 0.186 ± 0.073. Receiver operating characteristics curve analysis showed an absorbance cut-off value of 0.407. One negative serum gave an absorbance outside the normal range, giving a specificity of 99.2%. For the 12 sera from falcons with confirmed aspergillosis, nine gave absorbance values ≥ cut-off, giving a sensitivity of 75%. The Afmp1p-based ELISA is useful for serodiagnosis of falcons with aspergillosis.

An In Vitro Study on the Effects of Post-Core Design and Ferrule on the Fracture Resistance of Endodontically Treated Maxillary Central Incisors.

BACKGROUND: Endodontically treated teeth have significantly different physical and mechanical properties compared to vital teeth and are more prone to fracture. The study aims to compare the fracture resistance of endodontically treated teeth with and without post reinforcement, custom cast post-core and prefabricated post with glass ionomer core and to evaluate the ferrule effect on endodontically treated teeth restored with custom cast post-core. MATERIALS AND METHODS: A total of 40 human maxillary central incisors with similar dimensions devoid of any root caries, restorations, previous endodontic treatment or cracks were selected from a collection of stored extracted teeth. An initial silicone index of each tooth was made. They were treated endodontically and divided into four groups of ten specimens each. Their apical seal was maintained with 4 mm of gutta-percha. Root canal preparation was done and then post core fabrication was done. The prepared specimens were subjected to load testing using a computer coordinated UTM. The fracture load results were then statistically analyzed. One-way ANOVA was followed by paired t-test. RESULTS: 1. Reinforcement of endodontically treated maxillary central incisors with post and core, improved their fracture resistance to be at par with that of endodontically treated maxillary central incisor, with natural crown. 2. The fracture resistance of endodontically treated maxillary central incisors is significantly increased when restored with custom cast post-core and 2 mm ferrule. CONCLUSION: With 2 mm ferrule, teeth restored with custom cast post-core had a significantly higher fracture resistance than teeth restored with custom cast post-core or prefabricated post and glass ionomer core without ferrule.