Hari Mohan Saxena

Isolation of a bacteriophage against Salmonella Dublin and determination of its physical resistance under varied in vitro conditions

°C and direct sunlight beyond 5 days was found to be deleterious for survival of the phage.

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.

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.

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.

A New Superagglutination Test to Minimize False Negative and False Positive Results Common with Plate/Slide Agglutination Tests for the Diagnosis of Infectious Diseases

Agglutination is the clumping of antigenic particles by antibodies. Several diagnostic kits for infectious diseases of animals and humans, based on slide agglutination/plate agglutination tests like the Rose Bengal Plate Test ( RBPT) for diagnosis of brucellosis are used worldwide. False negative and false positive results are commonly encountered in these conventional agglutination tests. Simple, cost effective modifications can help in circumventing these problems. The novelSuperagglutinationTest reported here is a modified slide / plate agglutination test. False negative results due to smaller clumps formed by low titer of antibodies in serum are minimized by the addition of biotinylated antiglobulin followed by Avidin which forms easily detectable larger clumps. Similarly, prior staining of serum antibodies with a dye helps in differentiating a specific agglutinate formed by both antigen and antibody, from a non -specific aggregate of antigen alone that leads to false positive results.Superagglutinationis more sensitive than the current agglutination tests and agglutination based diagnostic kits. The modification steps are easy to perform and give a more accurate diagnosis without much increase in the cost of

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 new bacteriophage based luminescence assay for diagnosis of brucellosis

Brucellosis is a highly contagious zoonotic disease for which accurate and cost effective pen side diagnostics are not available. Here, we explored application of bacteriophage in developing a pen side diagnostic assay for Brucellosis. We have isolated a lytic brucellaphage and exploited the phenomena of ATP release on phage mediated lysis of bacteria and ATP catalyzed luciferase luciferin reaction to develop a novel assay for diagnosis of Brucellosis from clinical samples. Mean luminescence was 1616.333 + 662.608 for Brucella positive uterine secretions alone and 18507 + 3327.018 for phage treated positive samples. There was a significant difference (P <0.01) between the two values. The average increase in luminescence was 10.03 folds. The mean luminescence of negative samples treated with phage was 700.333 + 364.664. The difference between the luminescence of phage treated positive and negative samples was significant (P <0.01). The positive/negative status of animals from which the samples were derived was confirmed by Rose Bengal Plate Test and ELISA. The novel assay is simple, easy, accurate and field applicable assay for Brucellosis.T current investigation was carried out to evaluate oxidative stress serum biomarkers and antioxidant concentrations in pneumonic goat. Thirty goats of different age and sex belonging to private farms in Giza governorate were used in this study. All goats were exposed to complete and comprehensive clinical examination (rectal temperature, pulse and respiratory rates) which revealed significant increase (p≤0.05) in pneumonic goats. The animals were divided into two equal groups: Apparently healthy (15), diseased (15). Blood samples were collected from both groups in clean test tubes to determine various serum biomarkers as enzymatic activates of superoxidase dismutase (SOD), glutathione peroxidase (GPX) in erythrocyte haemolysate, catalase (CAT), vitamin C, vitamin E and selected biochemical parameters as (Albumin (Alb), aspartate aminotransaminase (AST), alanine aminotransaminase (ALT), urea and creatinine). The levels of superoxidase dismutase, glutathione peroxidase were significant higher (p≤0.05) in pneumonic goats than healthy while vitamins C, E were significant decrease (p≤0.05) in pneumonic goats. Serum biochemical analysis revealed significant decrease (p≤0.05) in Albumin with significant increase (p≤0.05) in ALT, AST and creatinine levels in pneumonic goats. It can be concluded that pneumonia served to produce oxidative stress in the goat with increased activities of antioxidant enzymes to facing excessive production of free radicals. For healthier status in small ruminant antioxidant supplementation is very important as supportive treatment in diseased cases and preventive aid in healthy cases. Antioxidants play a critical role in protection of animal from oxidative stress and ensuring rapid curing in diseased cases.

A Cohort Study on Some Immunohematological Parameters in Brucellosis Affected Cattle

Little is known about host immunodynamics during Brucellosis. We conducted immunohematological study in Brucellosis affected cattle for 3 months. Serum proteins and globulins were decreased very significantly (P<0.01) from zero day (0D) to 90D and significantly (P<0.05) from 0D to 30D. Globulins decreased significantly (P<0.05) from 0D to 75D. Titers of anti-Brucella antibodies by Standard Tube Agglutination Test, Microagglutination Test and Indirect Hemagglutination did not differ significantly among themselves from 0D to 90D. However, in ELISA, there was a very significant (p<0.01) increase in titers from 0Day to 60D, 75D and 90D; from 30Day to 60D, 75D and 90D; and from 45Day to 60D, 75D and 90D, respectively. Levels of leukocytes, lymphocytes and neutrophils did not differ significantly. However, monocyte levels increased from 45D to 60D and then decreased from 60D to 75D significantly (P<0.05). Data reflects the immunohematological dynamics in response to disease and provides insight for suitable intervention.

A simple innovative modification to enhance the efficacy of the complement fixation test.

Received: Revised: Accepted: 2013–11–07 2013–12–22 2013–12–23 The Complement Fixation Test (CFT) is a serodiagnostic test of high sensitivity commonly used in the diagnosis of infectious diseases like Brucellosis. However, the conventional CFT (cCFT) may sometimes give false negative or false positive result and may lead to incorrect diagnosis. If the results are not read within 30 minutes of finishing the test, it tends to show the signs of negativity making it difficult to find the true results. In the present study, serum samples from Brucellosis affected or healthy cattle were subjected to a modified CFT (mCFT) where, in addition to the regular procedure of the CFT, anti–hemolysin antiglobulin {i.e., [F(ab’)2] of antibody against rabbit IgG}, was added to facilitate the reading of the test. The innovative modification showed a clear hemolysis in negative samples as the antiglobulin binds to the haemolysin leading to pairing of the antibody molecules, enhancing the complement mediated haemolysis. The novel method could help in differentiating the positive and negative samples more explicitly and the test could be read more accurately. In the modified CFT, the results could be recorded easily even after 2 hours of finishing the test, minimizing the chances of wrong diagnosis. The modified CFT with a better readout would enable more accurate serodiagnosis of infectious diseases. All copyrights reserved to Nexus® academic publishers| The present study was conducted in Jind district of Haryana to ascertain the various sources of information is being utilized by the livestock farmers and to assess the extent of use of various sources for obtaining scientific livestock farming information. A multistage random sampling technique was followed for selection of two blocks, eight villages and 240 dairy farmers. The data was collected through pre-tested well-structured interview schedule and questionnaire. The study revealed that most of the dairy farmers obtained information from veterinary surgeon (93.75%), VLDA (90.88%), progressive farmers (83.75%), and experts from agricultural university (71.43%) regarding various aspects of breeding, feeding, health care and management practices. The study also revealed that most of the livestock farmers acquired the latest livestock information through their respective VLDAs followed by neighbours, friends, progressive livestock farmers and Veterinary Surgeons. Correlation analysis revealed that the sources of information were used by livestock farmers had positive and significant correlation with socio-economic status (SES), occupation, education of respondent, extension contact, mass media exposure, risk orientation and cosmopoliteness-localiteness. Age had negative correlation, while social participation, family education status and annual income were found positive but non-significant correlation with sources of information used by livestock farmers. The R2 value indicated that all the thirteen independent variables fitted in the regression equation had explained about 47 per cent variation towards various sources of information. Further, values of ‘t’ for ‘b’ in case of SES, occupation, social participation, herd size, mass media exposure, risk orientation and cosmopoliteness-localiteness were found to have positive and significant influence on sources of information used by dairy farmers.

New marker vaccine and DIVA assays for hemorrhagic septicemia in cattle

A R T I C L E I N F OC cancer is one of the most common gynecological cancers in world, but in India it is the most common cancer among women. Persistence infection with high-risk human papillomavirus (HR-HPV) is the most important risk factor. The sequence variation(s) in the most common HR-HPV type i.e. HPV-16 leads to altered biological functions with possible clinical significance in different geographical locations. Sixteen major variations (V1-V16) in full length L1 gene of HPV-16 were identified from cervical cancer tissue biopsies and showed their effect on immunogenicity. The effect of these major variations on the epitopes were predicted by in-silico methods and the immunogenicity of variant and respective reference DNA vaccine constructs were evaluated by administration of prepared DNA vaccine constructs in female BALB/c mice to evaluate antibody titer. In present study, L500F (V16) variation showed ~2.7 fold increase in antibody titer, whereas T379P (V8) showed ~0.4 fold decrease in antibody titer after final injection. These results showed a promising roadmap for the development of DNA based vaccine and for the generation of effective response need to study more prevalent variants of the HPV in the Indian population.L Pasteur coined the word “vaccine” to tribute first successful immunization against small pox by Edward Jenner. The term vaccine was innovated from “vacca” (cow) since Edward Jenner used cowpox virus to prevent smallpox infection. Vaccination is meant to active immunity in an individual, so that subsequent contact with the microorganism following natural infection induces strong protective immune response. Vaccine is a suspension of live or inactivated or fractionated bacteria or viruses which have been make non-pathogenic and is given to induce an immune response and prevent disease. Inactivated vaccines are produced by growing a virulent virus in eggs and then treating with an inactivating agent like beta propiolactone. Recombinant vaccines are which genes for desired antigens of a microbe are inserted into a vector. DNA is injected into the muscle of the animal being vaccinated with a gene gun that uses compressed gas to blow the DNA into the muscle cells. Live vaccines are easy to prepare and relatively inexpensive and give good immunity. Live vaccines are the heat resistant vaccines and they have the advantage in village use and easy transportation in villages. Finally cost is a significant factor to consider the preparation of vaccines. All the live vaccines are relatively cheap and can be even cheaper if they are produced locally. Inactivated vaccine is more expensive and the recombinant vaccines are very expensive when produced commercially. The selection of which vaccine to use is going to depend on the previous factors and on the particular regional conditions.C therapy has seen enormous growth in the last few decades in terms of new molecules and novel targeted formulations making a successful entry into the treatment paradigm. The semisynthetic taxane derivative, docetaxel holds great promise in treating solid tumours. However, owing to poor solubility of docetaxel in aqueous milieu, it is formulated as a solution in polysorbate 80, TaxotereO. The solubilizer (polysorbate 80) used, is associated with side effects like hypersensitivity reactions which demand co-administration of dexamethasone. This in turn, compromises immunity of cancer patients, often resulting in discontinuation of docetaxel treatment. The inability to target tumour tissues is another limitation of TaxotereO. In the light of the drawbacks associated with existing docetaxel therapy, the current study presents development of alternative formulations based on polymeric micelles fabricated using biodegradable polymers. Initially, polyethylene glycol-polycaprolactone amphiphilic copolymers of different molecular weights were synthesized by ring opening bulk polymerization and the products characterized (through IR, H1-NMR, XRD, DSC and measurement of critical micelle concentrations). These, in turn, were employed in the preparation of micellar systems containing solubilized docetaxel. The resultant micellar dispersions were characterized for particle size and zeta potential, drug content and efficiency of entrapment of docetaxel, morphology by TEM and for in vitro release studies. The biodegradable copolymers used to fabricate these systems were found to possess low CMC values, thereby conferring resistance to breakdown on dilution encountered in bloodstream in vivo. Also, in vitro cytotoxicity as evaluated in non-small cell lung cancer cell line A549 demonstrated that simple and mixed micelles were as effective as docetaxel solution and bore potential to be used as substituents for polysorbate 80 based formulations.