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.

Introductioncompared to foot and mouth disease, rinderpest, anthrax and black quarter, respectively [4]. The estimated Pasteurella multocida, a commensal of digestive annual economic losses in India due to Pasteurella and respiratory tracts of warm-blooded animals, causes multocida infections alone were found to be to the tune disease in animals (bovines, porcines, rabbits, and of Rs. 228 millions [5]. poultry) weakened by stresses such as viral infections, Humoral immunity plays an important role in heat, cold or humidity with aerosol transmission of protection against the disease. Vaccination has a infection between animals [1]. Haemorrhagic Septigreater effect in controlling mortality in HS than any caemia (HS) is an acute, fatal septicaemic disease of other measure [6]. The vaccines in use against HS cattle and buffaloes caused by the bacterium Pasteurella include broth bacterins, alum precipitated, aluminium multocida and occur as a catastrophic epizootic in hydroxide gel and oil adjuvant vaccines [7]. The most many Asian and African countries resulting in high widely used vaccines in Asia being the whole cell mortality and morbidity. Serotypes B: 2 and E: 2 of P.formalin killed P. multocida P bacterin precipitated 52 multocida are associated with hemorrhagic septicaewith alum or emulsified in aluminium hydroxide gel. mia in cattle and buffaloes in Asia and Central AfricaThe alum precipitated and the aluminium hydroxide respectively [2]. The interactions between host factors gel vaccines are reported to confer immunity for four to and certain specific bacterial virulence factors, like six months. Oil adjuvant vaccine that imparts higher lipopolysaccharides (LPS), capsule, outer membrane level of immunity (up to 1 year) poses difficulty in proteins (OMP), fimbrial protein etc are responsible for injecting owing to its viscous nature and it induces the pathogenesis of the disease [3].inflammation at the site of injection making it In India, HS has been identified as number one unpopular among the field users [8]. Although, killer disease of bacterial origin among cattle and vaccination against HS in endemic areas with currently buffaloes. In a retrospective analysis of disease occurrence available vaccines is conducted regularly, outbreaks in cattle and buffaloes in India during the years 1974-still occur emphasizing the need for improving the 1986, it was found that HS was responsible for the immunogenicity of the currently available vaccine highest mortality and the second highest morbidity rate preparations [9]. Microtiter Agglutination Test (MAT), Indirect Haemaaglutination Assay (IHA) and Enzyme Linked Immunosorbent Assay (ELISA) are usually employed to detect serum antibody levels in immunized Abstract Aim: The present study was carried out in 100 cattle to assess the antibody response to Haemorrhagic Septicaemia alum precipitated vaccine by Microtiter Agglutination Test (MAT), Indirect Haemaaglutination Assay (IHA) and Monoclonal Antibody based Indirect Enzyme Linked Immunosorbent Assay (ELISA). Mater...

Journal homepage: http://www.ijcmas.com Brucellosis is an important zoonotic disease globally that causes huge economic losses to the livestock owners and is of public health significance. Brucellosis in animals is endemic in India. In sheep and goats, Brucellosis is mainly caused by Brucella melitensis whereas Brucella ovis causes the disease in sheep. The symptoms in infected sheep and goats are abortions, stillbirths and the birth of weak offsprings. Animals that abort may retain the placenta. Sheep and goats usually abort only once, but reinvasion of the uterus and shedding of organisms can occur during subsequent pregnancies. Several studies have been carried out on seroepidemiology of caprine and ovine Brucellosis in India. The tests commonly used for diagnosis of Brucellosis are the milk ring test, Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (STAT), Microtiter Plate Agglutination Test (MAT) and ELISA. The RBPT is a rapid screening test for the diagnosis of Brucellosis. The sensitivity of RBPT is very high (>99%) but the specificity can be low and it could sometimes give a false positive result. Its positive predictive value is low and a positive test result requires confirmation by a more specific test. Isolation and culture of Brucella organisms is the gold standard test for the diagnosis of Brucellosis. K e y w o r d sOvine Brucellosis, Caprine Brucellosis, Serodiagnosis, Seroepidemiology

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ObjectiveThe 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).MethodThe 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.ResultThe 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.ConclusionThe 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.