Immunization Of Rabbits Research Articles

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells1,2. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.

Characterization of humoral and cell-mediated immunity in rabbits orally infected with Encephalitozoon cuniculi

Encephalitozoonosis is a common infectious disease widely spread among rabbits. Encephalitozoon cuniculi, is considered as a zoonotic and emerging pathogen capable of infecting both immunocompetent and immunocompromised hosts. The aim of the study was to describe in detail the spread of the E. cuniculi in a rabbit organism after experimental infection and the host humoral and cellular immune response including cytokine production. For that purpose, healthy immunocompetent rabbits were infected orally in order to simulate the natural route of infection and euthanised at 2, 4, 6 and 8-weeks post-infection. Dissemination of E. cuniculi in the body of the rabbit was more rapid than previously reported. As early as 2 weeks post-infection, E. cuniculi was detected using immunohistochemistry not only in the intestine, mesenteric lymph nodes, spleen, liver, kidneys, lungs and heart, but also in nervous tissues, especially in medulla oblongata, cerebellum, and leptomeninges. Based on flow cytometry, no conspicuous changes in lymphocyte subpopulations were detected in the examined lymphoid organs of infected rabbits. Cell-mediated immunity was characterized by ability of both CD4+ and CD8+ T cells to proliferate after stimulation with specific antigens. Th1 polarization of immune response with a predominance of IFN-γ expression was detected in spleen, mesenteric lymph nodes and Peyer’s patches. The increased expression of IL-4 and IL-10 mRNA in mixed samples from the small intestine is indicative of balanced control of IFN-γ, which prevents tissue damage. On the other hand, it can enable E. cuniculi to survive and persist in the host organism in a balanced host-parasite relationship. The Th17 immunity lineage seems to play only a minor role in E. cuniculi infection in rabbits.

Enzyme immunoassay of vitamin d3 metabolites

We report herein improved version of the synthesis of hapten based on cholecalciferol and its active metabolite 25-hydroxycholecalcalferol. The methodology of obtaining high-molecular immunogenic conjugates of vitamin D3 derivatives with bovine serum albumin and horseradish peroxidase conjugates for direct ELISA was optimized. Immunisation of rabbits was carried out and polyclonal antibodies to 25-hydroxycholecalceferol were obtained and tested in an enzyme-linked immunosorbent assay. To improve the accuracy of the method, the sample preparation procedure was optimized, including the release of vitamin D3 and its active metabolites from complexes with vitamin D-binding protein.

An Isoform of the Oncogenic Splice Variant AIMP2-DX2 Detected by a Novel Monoclonal Antibody.

AIMP2-DX2, an exon 2-deleted splice variant of AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein 2), is highly expressed in lung cancer and involved in tumor progression in vivo. Oncogenic function of AIMP2-DX2 and its correlation with poor prognosis of cancer patients have been well established; however, the application of this potentially important biomarker to cancer research and diagnosis has been hampered by a lack of antibodies specific for the splice variant, possibly due to the poor immunogenicity and/or stability of AIMP2-DX2. In this study a monoclonal antibody, H5, that specifically recognizes AIMP2-DX2 and its isoforms was generated via rabbit immunization and phage display techniques, using a short peptide corresponding to the exon 1/3 junction sequence as an antigen. Furthermore, based on mutagenesis, limited cleavage, and mass spectrometry studies, it is also suggested that the endogenous isoform of AIMP2-DX2 recognized by H5 is produced by proteolytic cleavage of 33 amino acids from N-terminus and is capable of inducing cell proliferation similarly to the uncleaved protein. H5 monoclonal antibody is applicable to enzyme-linked immunosorbent assay, immunoblot, immunofluorescence, and immunohistochemistry, and expected to be a valuable tool for detecting AIMP2-DX2 with high sensitivity and specificity for research and diagnostic purposes.

Phage Display Detection of Mimotopes that Are Shared Epitopes of Clinically and Epidemiologically Relevant Enterobacteria

Background: Escherichia coli and Salmonella are etiologic agents of intestinal infections. A previous study showed the presence of shared epitopes between lipopolysaccharides (LPSs) of E. coli O157 and Salmonella. Aim: Using phage display, the aim of this study is to identify mimotopes of shared epitopes in different enterobacterial LPSs. Methods: We use anti-LPS IgG from E. coli O157 and Salmonella to select peptide mimotopes of the M13 phage. The amino acid sequence of the mimotopes is used to synthesize peptides, which are in turn used to immunize rabbits. The antibody response of the resulting sera against the LPSs and synthetic peptides (SPs) is analyzed by ELISA and by Western blot assays, indicating that LPS sites are recognized by the same antibody. In a complementary test, the reactions of human serum samples obtained from the general population against the SPs and LPSs are also analyzed. Results: From the last biopanning phase, sixty phagotopes are selected. The analysis of the peptide mimotope amino acid sequences shows that in 4 of them the S/N/A/PF motif is a common sequence. Antibodies from the sera of immunized rabbits with SP287/3, SP459/1, SP308/3, and SP073/14 react against both their own peptide and the different LPSs. The Western blot test shows a sera reaction against both the lateral chains and the cores of the LPSs. The analysis of the human sera shows a response against the SPs and LPSs. Conclusion: The designed synthetic peptides are mimotopes of LPS epitopes of Salmonella and E. coli that possess immunogenic capacity. These mimotopes could be considered for use in the design of vaccines against both enterobacteria.

Positive Selection of Specific Antibodies Produced against Fusion Proteins

A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography.

Immunochemical Detection of Protein Modification Derived from Metabolic Activation of 8-Epidiosbulbin E Acetate.

Furanoid 8-epidiosbulbin E acetate (EEA) is one of the most abundant diterpenoid lactones in herbal medicine Dioscorea bulbifera L. (DB). Our early work proved that EEA could be metabolized to EEA-derived cis-enedial (EDE), a reactive intermediate, which is required for the hepatotoxicity observed in experimental animals exposed to EEA. Also, we found that EDE could modify hepatic protein by reaction with thiol groups and/or primary amines of protein. The present study was inclined to develop polyclonal antibodies to detect protein modified by EDE. An immunogen was prepared by reaction of EDE with keyhole limpet hemocyanin (KLH), and polyclonal antibodies were raised in rabbits immunized with the immunogen. Antisera collected from the immunized rabbits demonstrated high titers evaluated by enzyme-linked immunosorbent assays (ELISAs). Immunoblot analysis showed that the polyclonal antibodies recognized EDE-modified bovine serum albumin (BSA) in a hapten load-dependent manner but did not cross-react with native BSA. Competitive inhibition experiments elicited high selectivity of the antibodies toward EDE-modified BSA. The antibodies allowed us to detect and enrich EDE-modified protein in liver homogenates obtained from EEA-treated mice. The developed immunoprecipitation technique, along with mass spectrometry, enabled us to succeed in identifying multiple hepatic proteins of animals given EEA. We have successfully developed polyclonal antibodies with the ability to recognize EDE-derived protein adducts, which is a unique tool for us to define the mechanisms of toxic action of EEA.

Prediction, mapping and validation of tick glutathione S-transferase B-cell epitopes

In search of ways to address the increasing incidence of global acaricide resistance, tick control through vaccination is regarded as a sustainable alternative approach. Recently, a novel cocktail antigen tick-vaccine was developed based on the recombinant glutathione S-transferase (rGST) anti-sera cross-reaction to glutathione S-transferases of Rhipicephalus appendiculatus (GST-Ra), Amblyomma variegatum (GST-Av), Haemaphysalis longicornis (GST-Hl), Rhipicephalus decoloratus (GST-Rd) and Rhipicephalus microplus (GST-Rm). Therefore, the current study aimed to predict the shared B-cell epitopes within the GST sequences of these tick species. Prediction of B-cell epitopes and proteasomal cleavage sites were performed using immunoinformatics algorithms. The conserved epitopes predicted within the sequences were mapped on the homodimers of the respective tick GSTs, and the corresponding peptides were independently used for rabbit immunization experiments. Based on the dot blot assay, the immunogenicity of the peptides and their potential to be recognized by corresponding rGST anti-sera raised by rabbit immunization in a previous work were investigated. This study revealed that the predicted conserved B-cell epitopes within the five tick GST sequences were localized on the surface of the respective GST homodimers. The epitopes of GST-Ra, GST-Rd, GST-Av, and GST-Hl were also shown to contain a seven residue-long peptide sequence with no proteasomal cleavage sites, whereas proteasomal digestion of GST-Rm was predicted to yield a 4-residue fragment. Given that a few proteasomal cleavage sites were found within the conserved epitope sequences of the four GSTs, the sequences could also contain a T-cell epitope. Finally, the peptide and rGST anti-sera reacted against the corresponding peptide, confirming their immunogenicity. These data support the claim that the rGSTs, used in the previous study, contain conserved B-cell epitopes, which elucidates why the rGST anti-sera cross-reacted to non-homologous tick GSTs. Taken together, the data suggest that the B-cell epitopes predicted in this study could be useful for constituting epitope-based GST tick vaccines.

A Bivalent, Spherical Virus-Like Particle Vaccine Enhances Breadth of Immune Responses against Pathogenic Ebola Viruses in Rhesus Macaques.

The 2013-2016 Ebola outbreak in West Africa led to accelerated efforts to develop vaccines against these highly virulent viruses. A live, recombinant vesicular stomatitis virus-based vaccine has been deployed in outbreak settings and appears highly effective. Vaccines based on replication-deficient adenovirus vectors either alone or in combination with a multivalent modified vaccinia Ankara (MVA) Ebola vaccine also appear promising and are progressing in clinical evaluation. However, the ability of current live vector-based approaches to protect against multiple pathogenic species of Ebola is not yet established, and eliciting durable responses may require additional booster vaccinations. Here, we report the development of a bivalent, spherical Ebola virus-like particle (VLP) vaccine that incorporates glycoproteins (GPs) from Zaire Ebola virus (EBOV) and Sudan Ebola virus (SUDV) and is designed to extend the breadth of immunity beyond EBOV. Immunization of rabbits with bivalent Ebola VLPs produced antibodies that neutralized all four pathogenic species of Ebola viruses and elicited antibody-dependent cell-mediated cytotoxicity (ADCC) responses against EBOV and SUDV. Vaccination of rhesus macaques with bivalent VLPs generated strong humoral immune responses, including high titers of binding, as well as neutralizing antibodies and ADCC responses. VLP vaccination led to a significant increase in the frequency of Ebola GP-specific CD4 and CD8 T cell responses. These results demonstrate that a novel bivalent Ebola VLP vaccine elicits strong humoral and cellular immune responses against pathogenic Ebola viruses and support further evaluation of this approach as a potential addition to Ebola vaccine development efforts.IMPORTANCE Ebola outbreaks result in significant morbidity and mortality in affected countries. Although several leading candidate Ebola vaccines have been developed and advanced in clinical testing, additional vaccine candidates may be needed to provide protection against different Ebola species and to extend the durability of protection. A novel approach demonstrated here is to express two genetically diverse glycoproteins on a spherical core, generating a vaccine that can broaden immune responses against known pathogenic Ebola viruses. This approach provides a new method to broaden and potentially extend protective immune responses against Ebola viruses.

Preparation and in vivo evaluation of glyco-gold nanoparticles carrying synthetic mycobacterial hexaarabinofuranoside.

A number of bacterial glycans are specific markers for the detection and the serological identification of microorganisms and are also widely used as antigenic components of vaccines. The use of gold nanoparticles as carriers for glyco-epitopes is becoming an important alternative to the traditional conjugation with proteins and synthetic polymers. In this study, we aimed to prepare and evaluate in vivo glyco-gold nanoparticles (glyco-GNPs) bearing the terminal-branched hexaarabinofuranoside fragment (Ara6) of arabinan domains of lipoarabinomannan and arabinogalactan, which are principal polysaccharides of the cell wall of Mycobacterium tuberculosis, the causative agent of tuberculosis. In particular, we were interested whether the antibodies generated against Ara6-GNPs would recognize the natural saccharides on the cell surface of different mycobacterial strains. Two synthetic Ara6 glycosides with amino-functionalized spacer aglycons differing in length and hydrophilicity were directly conjugated with spherical gold nanoparticles (d = 15 nm) to give two sets of glyco-GNPs, which were used for the immunization of rabbits. Dot assays revealed cross-reactions between the two obtained antisera with the hexaarabinofuranoside and the 2-aminoethyl aglycon used for the preparation of glyco-GNPs. Both antisera contained high titers of antibodies specific for Mycobacteria as shown by enzyme-linked immunosorbent assay using M. bovis and M. smegmatis cells as antigens while there was only a weak response to M. phlei cells and no interaction with E. coli cells. The results obtained suggest that glyco-GNPs are promising agents for the generation of anti-mycobacterial antibodies.

Antibody response of a particle-inducing, liposome vaccine adjuvant admixed with a Pfs230 fragment

Pfs230 is a malaria transmission-blocking antigen candidate, expressed on the surface of Plasmodium falciparum gametocytes. A recombinant, his-tagged Pfs230 fragment (Pfs230C1; amino acids 443–731) formed serum-stable particles upon incubation with liposomes containing cobalt-porphyrin-phospholipid (CoPoP). In mice, immunization with Pfs230C1, admixed with the adjuvants Alum, Montanide ISA720 or CoPoP liposomes (also containing synthetic monophosphoryl lipid A; PHAD), resulted in elicitation of IgG antibodies, but only those induced with CoPoP/PHAD or ISA720 strongly reduced parasite transmission. Immunization with micrograms of Pfs230C1 adjuvanted with identical liposomes lacking cobalt (that did not induce particle formation) or Alum was less effective than immunization with nanograms of Pfs230C1 with CoPoP/PHAD. CoPoP/PHAD and ISA720 adjuvants induced antibodies with similar Pfs230C1 avidity but higher IgG2-to-IgG1 ratios than Alum, which likely contributed to enhanced functional activity. Unlike prior work with another transmission-blocking antigen (Pfs25), Pfs230C1 was found to be effectively taken up by antigen-presenting cells without particle formation. The anti-Pfs230C1 IgG response was durable in mice for 250 days following immunization with CoPoP/PHAD, as were antibody avidity and elevated IgG2-to-IgG1 ratios. Immunization of rabbits with 20 µg Pfs230C1 admixed with CoPoP/PHAD elicited antibodies that inhibited parasite transmission. Taken together, these results show that liposomes containing CoPoP and PHAD are an effective vaccine adjuvant platform for recombinant malaria transmission blocking antigens.

Effect of Rabbit Immunization with Yeast Antigens on the Activity of the Fraction of Serum Antimicrobial Peptides

The aim was to study the effect of rabbit immunization with different yeasts cells on overall antimicrobial serum activity and specific activity of antimicrobial peptides fraction.Material and methods. Rabbits were immunized with cells of Candida albicans, Rhodotorula mucilaginosa, Malassezia furfur, Cryptococcus neoformans, Geotrichum candidum, Trichosporon cutaneum and Saccharomyces cerevisiae. AMP fractions were obtained by filtration through 100 kDa filters. Overall activities of sera and their AMP-fractions were estimated spectrophotometrically.Results. Overall serum antimicrobial activity had tendency to decrease after immunization independently from yeast species. Activity of AMP-fractions after immunization with C. albicans, Cr. neoformans, G. candi dum и S. ce revisiae increased, but with other yeasts — decreased. The highest sensitivity to AMP fraction cor res ponded to C. albicans, especially for serum of the rabbit immunized with this yeasts — 1,5-fold higher com pare to control. Serum albumin concentration showed the positive correlation with overall antimicrobial activity against C. albicans (r = 0,760), R. mucilaginosa (r = 0,728), M. furfur (r = 0,723) и T. cutaneum (r = 0,588).Discussion. The results confirm our earlier data. Moreover, they expand current views on the effect of yeasts antigens on activity of serum AMP against different yeasts.Conclusion. Serum albumin and serum AMP demonstrated the specificity toward different yeasts: maximal activity they showed towards specia which more often play opportunistic role.

Effect of Rabbit Immunization with Yeast Antigens on the Activity of the Fraction of Serum Antimicrobial Peptides

The aim was to study the effect of rabbit immunization with different yeasts cells on overall antimicrobial serum activity and specific activity of antimicrobial peptides fraction.Material and methods. Rabbits were immunized with cells of Candida albicans, Rhodotorula mucilaginosa, Malassezia furfur, Cryptococcus neoformans, Geotrichum candidum, Trichosporon cutaneum and Saccharomyces cerevisiae. AMP fractions were obtained by filtration through 100 kDa filters. Overall activities of sera and their AMP-fractions were estimated spectrophotometrically.Results. Overall serum antimicrobial activity had tendency to decrease after immunization independently from yeast species. Activity of AMP-fractions after immunization with C. albicans, Cr. neoformans, G. candi dum и S. ce revisiae increased, but with other yeasts — decreased. The highest sensitivity to AMP fraction cor res ponded to C. albicans, especially for serum of the rabbit immunized with this yeasts — 1,5-fold higher com pare to control. Serum albumin concentration showed the positive correlation with overall antimicrobial activity against C. albicans (r = 0,760), R. mucilaginosa (r = 0,728), M. furfur (r = 0,723) и T. cutaneum (r = 0,588).Discussion. The results confirm our earlier data. Moreover, they expand current views on the effect of yeasts antigens on activity of serum AMP against different yeasts.Conclusion. Serum albumin and serum AMP demonstrated the specificity toward different yeasts: maximal activity they showed towards specia which more often play opportunistic role.

Engineered protein containing crotoxin epitopes induces neutralizing antibodies in immunized rabbits

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

Longitudinal Human Antibody Repertoire against Complete Viral Proteome from Ebola Virus Survivor Reveals Protective Sites for Vaccine Design

Evolution of antibody repertoire against the Ebola virus (EBOV) proteome was characterized in an acutely infected patient receiving supportive care alone to elucidate virus-host interactions over time. Differential kinetics are observed for IgM-IgG-IgA epitope diversity, antibody binding, and affinity maturation to EBOV proteins. During acute illness, antibodies predominate to VP40 and glycoprotein (GP). At day 13 of clinical illness, a marked increase in antibody titers to most EBOV proteins and affinity maturation to GP is associated with rapid decline in viral replication and illness severity. At one year, despite undetectable virus,a diverse IgM repertoire against VP40 and GP epitopes is observed suggesting occult viral persistence. Rabbit immunization experiments identify key immunodominant sites of GP, while challenge studies in mice found these epitopes induce EBOV-neutralizing antibodies and protect against lethal EBOV challenge. This study reveals markers of viral persistence and provides promising approaches for development and evaluation of vaccines and therapeutics.

Effect of lactic acid bacteria on Listeria monocytogenes infection and innate immunity in rabbits

The present study aimed to investigate the effect of probiotic lactic acid bacteria (LAB) addition on Listeria monocytogenes translocation and its toxin listeriolysin O (LLO), proinflammatory factors, immune organ indexes and serum immunoglobulins in farmed rabbits. Five treatments included negative control (NC), positive control (PC) with L. monocytogenes infection and supplemental LAB at 3.0 × 10<sup>6 </sup>(low-LAB, L-LAB), 3.0 × 10<sup>8</sup> (medium-LAB, M-LAB) and 3.0 × 10<sup>10 </sup>(high-LAB, H-LAB) CFU/kg of diet, respectively. The LAB was a mixture of equal amounts of Lactobacillus acidophilus (ACCC11073), Lactobacillus plantarum (CICC21863) and Enterococcus faecium (CICC20430). A total of 180 weaned rabbits (negative for L. monocytogenes) were randomly assigned to 5 groups with 6 replicates of 6 rabbits each in response to the 5 treatments. L. monocytogenes infection occurred on the first day of feeding trial and dietary LAB supplementation lasted for 14 days. The results showed that on days 7 and 14 post administration, L. monocytogenes in caecum, liver, spleen and lymph nodes was reduced in M-LAB and H-LAB compared to PC (P < 0.05), and linear and quadratic reducing trends were found in liver on day 7 (P ≤ 0.002). On day 14, mucosa LLO mRNA expression and serum TNFα, IL1β and IFNγ were reduced in the three LAB treatments (P < 0.05), and linear and quadratic trends were found on TNFα and IL1β (P ≤ 0.025); indexes of thymus and spleen, serum IgA and IgG were increased in the LAB treatments (P < 0.05). It is concluded that LAB can be used to alleviate L. monocytogenes infection and to improve the immune function of farmed animals.

Protection Efficacy of Oral Bait Probiotic Vaccine Constitutively Expressing Tetravalent Toxoids against Clostridium perfringens Exotoxins in Livestock (Rabbits)

Clostridium perfringens is an opportunistic pathogen. Its main virulence factors are exotoxins, which are the etiological agents of enteritis necroticans and enterotoxemia caused in livestock (cattle, sheep, and rabbits). Here, we demonstrated effective immune protection for rabbits against α, β, and ε exotoxins of C. perfringens provided by an oral tetravalent bait probiotic vaccine delivering α, ε, β1, and β2 toxoids of C. perfringens. Results showed that the recombinant probiotic had good segregational stability and good colonization ability in the rabbit intestinal tract. Oral administration of the probiotic vaccine can effectively elicit significant levels of antigen-specific mucosa sIgA and sera IgG antibodies with exotoxin-neutralizing activity. Additionally, oral immunization with the probiotic vaccine effectively promoted lymphoproliferation and Th1/Th2-associated cytokine production. The protection rate of immunized rabbits with the probiotic vaccine was 80% after challenging rabbits with a combination of C. perfringens (toxinotypes A, C, and D) and exotoxin mixture, which was better than the 60% provided by a commercial inactivated C. perfringens A, C, and D trivalent vaccine. Moreover, obvious histopathological changes were observed in the intestinal tissues of rabbits in the commercial vaccine and PBS groups. The bait probiotic vaccine can provide effective protection against C. perfringens exotoxins, suggesting a promising C. perfringens vaccination strategy.

Immunohistochemical, histological and ultrastructural evaluation of protection provided by cholera vaccine against V. cholerae O139

In our previous study, complete protection was observed in rabbit immunized with 1 × 1010 CFU of live attenuated VCUSM21P vaccine against challenge with 1 × 109 CFU Vibrio cholerae O139. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological, immunohistochemical and ultrastructural techniques. Severe pathology is evident in wild type injected ileum in non-immunized, showing extensive villous destruction, edema, necrosis and inflammation with infiltration of large numbers of inflammatory cells, extensive damage to the villi and microvilli with pore formation. Histology of ileum injected with wild type in immunized rabbit shows no significant pathological changes except for a few inflammatory cells in lamina propria with mild edema in mucosa and submucosa. immunohistochemical staining revealed O139 antigens of wild type are seen in the lamina propria of edematous villi, muscularis mucosa and submucosa with weak presence in the muscle coat in non-immunized rabbit after challenged with wild type in non-immunized rabbits, but in immunized rabbit localisation of the O139 LPS antigen is seen at the tips of the intact villi, within lamina propria and muscularis mucosa only. These observations suggest that the vaccine can effectively protect animals from any pathologic changes and eliminate V. cholerae O139 from the immunized animals.

STANDARDIZATION AND EVALUATION OF ANTIBODY RESPONSE BY PLATE-ELISA FOR THE DETECTION OF PARAMPHISTOMOSIS IN RUMINANTS

Disease transmission, diagnosis and preventive measures have become a major concern for the scientists working in national and international institution throughout the world. Paramphistomosis is a parasitic disease occurring in domestic ruminants causing economic loss to livestock industry. Early diagnosis of the disease is very important so that the loss due to disease can be curtailed by the appropriate treatment. Immunodiagnostic assays are helpful in the diagnosis of parasitic diseases. Hence immunological tests especially microtitre plate enzyme linked immunosorbant assay (ELISA) are the mainstay of diagnosis. Enzyme linked immunosorbant assay (ELISA), one of the immunodiagnostic assays is helpful in the diagnosis of paramphistomosis. More than 500 clinical/field sera samples of buffaloes, goats, sheep and cattle were collected from Bareilly, Delhi, Dehradun and Ludhiana under DST (GOI) sponsored project. Microtitre plate (Greiner) containing 96 well for Indirect Plate-ELISA was standardized for the detection of anti-Paramphistomunepiclitum antibodies in immunized rabbit using somatic antigen of P.epiclitum ranging from 1µg/ml to 10µg/ml, HRPO conjugate dilutions 1:1000 to 1:8000 and the range of sera dilution from 1:50 to 1:1,60,000. The optimum concentration of adult somatic P. epiclitum antigen was observed to be 2µg/ml, conjugate dilution 1:1000 and sera dilution at 1:200 and 1:400. A total of 222 clinical/field sera samples of ruminants were tested. Out of the total 106 ruminant samples were found to be positive with the incidence rate of 47.75 %. The highest percent positivity (84.0%) was found in naturally infected buffaloes followed by 26.25% in goats and 12.5% in sheep by indirect plate-ELISA. The observations on sensitivity and specificity of plate-ELISA test and immune response of antiP.epiclitum antibodies in experimental animal were also evaluated. The observation of high titre during the study is very effective for the detection of anti-P.epiclitum antibodies in field survey or in clinical cases. It is also helpful in the characterization of immunodominant antigens for the immunological control of the disease. Hence, indirect plateELISAis very important for the detection of paramphistomosis in domestic ruminants in early stages.

Reduction of Milk Contamination with Aflatoxin-M1 through Vaccination of Dairy Cattle with Aflatoxin-B1 Vaccine

Aflatoxin M1 is one of mycotoxin derivatives, which is secreted in milk of dairy cattle fed on feed contaminated with Aflatoxin-B1 (AFB1). The current study was designed to prepare a vaccine against AFB1and to evaluate its efficacy in reducing or preventing secretion of AFM1 in milk. Aflatoxin-B1 was prepared, purified and transformed into oxime, then it was fixed on bovine serum albumins. The AFB1-BSA conjugate was adjuvanted with Gold Nano particles then Montanide ISA 206. The prepared vaccine was used for immunization of rabbits by S/c routes as 100 µg/dose and dairy cattle by I/M routes as 500 µg/dose. The vaccinated animals were boosted at 3 weeks post primary immunization. Serum samples were collected and examined for the anti-AFB1 using AGPT. A mean titer of 15.2 AGPU/ml was detected at 2 weeks post primary vaccination then significantly increased till reached to 76.8 AGPU/ml at 6 weeks post Booster vaccination. All vaccinated rabbits were challenged with dose of 0.3 mg AFB1 toxin/Kg. The vaccinated rabbit showed 100% protection and no AFB1 toxin residue was detected in their livers. Milk samples were collected from non-vaccinated and AFB1-immunized dairy cattle then examined with ELISA for quantitation of AFM1 residues before and after vaccination. The results showed that the prepared AFB1 vaccine was safe, potent and able to reduce AFM1 release in milk of vaccinated heifers by 70%. So the vaccination of lactating animals with the AFB1vaccine might represent a valid tool for the prevention of AFM1 contamination of milk and dairy products.