Effect of Phage Targeting Therapy of Brucellosis on Host Antibody Response in Cattle.

Brucellosis caused by Brucella organisms is a major zoonosis globally. It causes heavy losses through abortions, delayed conception, and infertility in animals. Brucella is an intracellular bacterium. Antibiotic therapy for bovine Brucellosis is expensive and may sometimes be ineffective as Brucella can become resistant. Once infected, the animal may remain carrier and shed bacteria in milk, semen, and uterine discharges spreading infection to others for a long period. The live attenuated Brucella abortus strain S19 organisms that are commonly used as a vaccine were employed to deliver a broad acting lytic brucellaphage inside the phagocytes in vivo to reach the virulent Brucella hiding intracellularly. The phage pulsed vaccine induced sustained and significantly high titers of anti-Brucella antibodies compared with the untreated animals and animals vaccinated with S19 vaccine alone as estimated by standard tube agglutination test, microagglutination test, indirect hemagglutination assay test, and enzyme-linked immunosorbent assay. The current investigation is perhaps the first systematic attempt whereby efficacy of brucellaphage pulsed vaccine preparation in induction of specific antibody response was evaluated in cattle.

Efficacy of bacteriophage Lysed Pasteurella marker vaccine in laboratory animal models with a novel DIVA for haemorrhagic septicaemia.

OBJECTIVE: The present study aimed at evaluating the efficacy of an improved phage lysate marker vaccine for haemorrhagic septicaemia in mice and rabbit model and development of a DIVA ELISA based on iron restricted outer membrane protein (IROMP). METHOD: The experimental vaccine was prepared by lysing P. multocida B:2 grown under iron restricted conditions with a Pasteurella bacteriophage and addition of an alum adjuvant to enhance the immunogenicity. The vaccine was administered in mice and rabbits divided into two group each. Phage lysate vaccine (PL-VacI) was administered to group I mice and rabbits whereas group II mice and rabbits received alum precipitated HS vaccine (HS-VacII). Antibody titres were monitored 0, 30, 60, 90, 210 and 240 dpv. An IROMP (130 kDa) based indirect ELISA was also developed to differentiate between infected and vaccinated animals. The Pasteurella phage isolated in present study was sequenced at Georgia Genomic Facilty, Georgia. RESULT: The sequence of PMP-GAD-IND (Pasteurella bacteriophage) was deposited in GenBank under no KY203335. The group I mice and rabbits vaccinated with Phage lysate vaccine (PL-VacI) group revealed significantly higher antibody titres than group II mice and rabbits receiving alum-precipitated bacterin (HS-VacII) by MAT, IHA and ELISA (P < 0.05 and P < 0.001). The peak log 10 values (3.46) in case of group I mice by ELISA were attained at 90DPI whereas in group II mice the peak values at 90DPI were 2.82. Mean log10 titres by ELISA in group I and II rabbits were 2.43 and 2.35 respectively at 30DPI whereas at 120DPI the titres were 3.29 and 2.75, respectively. The DIVA ELISA detected presence of a novel 137 kDa IROMP/siderophore antibody in sera of group I mice and rabbits (PL-VacI) absent in sera of mice and rabbits given HS-VacII. CONCLUSION: The bacteriophage based marker vaccine (PL-VacI) had a more effective and longer immune response against HS in mice and rabbit in comparison to the widely used alum precipitated HS vaccine (HS-VacII). Moreover, the development of a recombinant IROMP based indirect ELISA could serve as an excellent tool to differentiate between infected and vaccinated cattle and buffaloes for effective control of HS.

A novel immunotherapy of Brucellosis in cows monitored non invasively through a specific biomarker.

Brucellosis is an important zoonotic disease causing huge economic losses worldwide. Currently no effective immunotherapy for Brucellosis or any biomarker to monitor the efficacy of therapy is available. Treatment is ineffective and animals remain carrier lifelong. S19 and RB51 are live attenuated vaccine strains of Brucella abortus. However, S19 induces only antibody, ineffective for intracellular pathogen. RB51 induces cell mediated immunity (CMI) but it is Rifampicin resistant. Both organisms are secreted in milk and can infect humans and cause abortions in animals. Phage lysed bacteria (lysates) retain maximum immunogenicity as opposed to killing by heat or chemicals. We report here the successful immunotherapy of bovine Brucellosis by phage lysates of RB51 (RL) and S19 (SL). The SL induced strong antibody response and RL stimulated CMI. In vitro restimulation of leukocytes from RL immunized cattle induced interferon gamma production. A single subcutaneous dose of 2 ml of cocktail lysate (both RL and SL), eliminated live virulent Brucella from Brucellosis affected cattle with plasma level of Brucella specific 223 bp amplicon undetectable by RT-PCR and blood negative for live Brucella by culture in 3 months post-immunization. This is the first report on minimally invasive monitoring of the efficacy of antibacterial therapy employing plasma RNA specific for live bacteria as a biomarker as well as on the use of RB51 phage lysate for successful immunotherapy of Brucellosis in cattle.

A comparison of titers of anti-Brucella antibodies of naturally infected and healthy vaccinated cattle by standard tube agglutination test, microtiter plate agglutination test, indirect hemagglutination assay, and indirect enzyme-linked immunosorbent assay.

AIM: We determined the antibody response in cattle naturally infected with brucellosis and normal healthy adult cattle vaccinated during calf hood with strain 19. MATERIALS AND METHODS: The antibody titers were measured by standard tube agglutination test (STAT), microtiter plate agglutination test (MAT), indirect hemagglutination assay (IHA), and indirect enzyme-linked immunosorbent assay (iELISA) as per standard protocols. RESULTS: The mean STAT titers were 1.963±0.345 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was extremely significant (p<0.0001). The mean MAT titers were 2.244±0.727 in infected cattle and 1.200±0.155 in healthy vaccinated cattle. The difference was very significant (p<0.005). The mean IHA titers in infected cattle were 2.284±0.574, and those in healthy vaccinated cattle were 1.200±0.155. The difference was extremely significant (p=0.0002). However, the difference in mean iELISA titers of infected cattle (1.3678±0.014) and healthy vaccinated cattle (1.367±0.014) was non-significant. The infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals. However, it cannot be ascertained whether these antibodies are due to vaccine or response to infection. Since the infected animals had been vaccinated earlier, the current infection may suggest that vaccination was unable to induce protective levels of antibody. The heightened antibody response after infection may also indicate a secondary immune response to the antigens common to the vaccine strain and wild Brucella organisms. CONCLUSION: The brucellosis infected animals showed very high titers of agglutinating antibodies compared to the vaccinated animals.

On genome annotation of Brucellaphage Gadvasu (BpG): discovery of ORFans for integrated systems biology approaches.

Brucellaphage Gadvasu (BpG) is a lytic phage infecting Brucella spp. Brucellaphages contain dsDNA as genetic material and are short-tailed particles with host-specificity. Here, we report the challenges on annotation in the complete genome sequence of BpG when compared with that of a recent broad host-range brucellaphage Pr, an original reference genome. The extracted DNA was subjected to genome sequencing with Illumina technology and assembled using SSAKE/Velvet. A significant number of genes were found to be similar between the phages with sequence analysis revealing conserved open reading frames that correspond to 33 gene ontology classifiers, transcriptional terminators and a few putative transcriptional promoters. The analyses revealed that the genome constitutes 1269 contigs and 275 genes encoding 260 proteins. The sequence comparison from the reference data indicated that the genome shares an approximately 70 % nucleotide similarity and differs mainly in the region encoding proteins. We bring this commentary providing an overview of how this exemplar genome can allow us to understand these known unknown regions in brucellaphages.

Simple solutions to false results with plate/slide agglutination tests in diagnosis of infectious diseases of man and animals.

We have developed a new Superagglutination test for serodiagnosis of infectious diseases. It differs from conventional plate/slide agglutination tests (PAT/SAT) by three additional steps: prior staining of serum antibody by adding a dye and addition of diluted biotinylated antiglobulin and avidin in sequence after mixing the antigen with the test serum. The new steps circumvent the problems of false positive and false negative results of PAT/SAT. In serodiagnosis of brucellosis, Superagglutination test had higher positive predictive value and specificity than Rose Bengal Plate Test (RBPT) and Standard Tube Agglutination Test (STAT) and higher negative predictive value and sensitivity than RBPT, STAT, ELISA and Complement Fixation Test (CFT).•Superagglutination is a simple, accurate and economic screening test for infections.•More specificity, sensitivity, positive & negative predictive value than RBPT, STAT.•More sensitivity, negative predictive value than ELISA and Complement Fixation Test.

Innovative modifications to Rose Bengal plate test enhance its specificity, sensitivity and predictive value in the diagnosis of brucellosis.

Current agglutination tests occasionally yield false results. Superagglutination test reduced false results, had higher sensitivity (95.88%) and negative predictive value (95.83%) than Rose Bengal plate test (RBPT), Standard Tube Agglutination test (STAT), ELISA, and Complement Fixation test and specificity (89.32%) and positive predictive value (89.42%) higher than RBPT and STAT.

Abrogation of DTH response and mitogenic lectin- and alloantigen-induced activation of lymphocytes by calcium inhibitors TMB-8 and BAPTA-AM.

In vitro treatment of mouse lymphocytes with an intracellular calcium chelator BAPTA-AM significantly decreased lectin Concanavalin-A (Con-A)-induced and mixed lymphocyte reaction (MLR) mediated, alloantigen-induced lymphocyte activation as indicated by decreased percentage of lymphoblasts among the BAPTA-treated lymphocytes. In vivo treatment of mice with intracellular Ca(2+) antagonist TMB-8 was found to substantially impair delayed type hypersensitivity (DTH; cell mediated immune) response, as indicated by decreased footpad swelling on tuberculin challenge of mice sensitized with BCG, after a single treatment with a low dose of 0.01mg of TMB-8 per mouse. Interestingly, a second injection of a higher dose of TMB-8 (0.1mg per mouse) resulted in very significant (p=0.001) abrogation of DTH as indicated by complete absence of swelling of foot pad after PPD challenge in BCG-primed treated mice. All mice in this group showed fully impaired DTH response. Lymphocytes of allosensitized mice gave a significantly higher (p<0.05) MLR response than the naïve mice. However, a single treatment of allosensitized mice with 0.1mg TMB-8 resulted into a lower MLR response, comparable in magnitude to that of untreated naïve mice.