Combinations of Toll-like receptor (TLR) agonists are being explored to alleviate systemic toxicity and achieve long-lasting desirable immune responses. In this study, we investigated the use of a triple-TLR agonists' combination (LPS, TLR4; R-848, TLR7 and CpG-ODN, TLR21) as an adjuvant with the inactivated Newcastle disease virus (NDV) vaccine in chickens. We assessed the immune response kinetics by stimulating chicken peripheral blood mononuclear cells (PBMCs) with the triple agonist combination and analyzing immune-related gene expression using quantitative PCR. Additionally, transcriptomic analysis was performed in spleen samples from birds injected with the triple TLR agonists. In the immunization study, birds were vaccinated with an inactivated NDV vaccine alone or in combination with different TLR agonist mixtures (single, dual, or triple), followed by assessment of both humoral and cellular immune responses and protection against challenge. Results from immune response study showed significant upregulation of IL-1β, IFN-γ, IL-4, IFN-β, iNOS and MHC-II transcripts, indicating the pro-inflammatory, anti-viral and mixed Th1/Th2 responses in triple TLR agonists' combination. Transcriptomic analysis revealed significant upregulation of 19 differentially expressed genes (DEGs) related to immune function in triple-TLR agonists signaling pathway such as pro-inflammatory, anti-inflammatory and anti-viral response. Furthermore, the immunization study demonstrated that the triple-TLR agonist combination, at a low dose exhibited no toxicity and significantly enhanced both humoral and cell-mediated immune responses, leading to higher antibody titres, increased T cell activation, and complete protection against a virulent NDV challenge. These findings suggest that the triple-TLR agonists' combination could improve vaccine efficacy and provide a cost-effective approach for vaccine formulations.

Combination of toll-like receptor (TLR) agonists is tried to minimize toxicity and obtain a desired immunological response at lower dosages. This study explores the effects of combining toll-like receptor (TLR) agonists, focusing on the interactions between TLR2 and TLR4 or TLR21 in chickens. We investigated the co-stimulatory effects of Pam3CSK4 (TLR2 agonist) with either lipopolysaccharide (LPS, TLR4 agonist) or CpG ODN (TLR21 agonist) on immune responses in the chicken peripheral blood mononuclear cells (PBMCs) and also in vivo. Co-stimulation of chicken PBMCs with Pam3CSK4 and LPS significantly upregulated IL-1β expression and synergistically enhanced the expression of Th1/Th2 cytokines (IL-12p40, IFN-γ, IL-4, IL-13), IL-10, IFN-β, iNOS genes, and NO production. Similarly, the combination of Pam3CSK4 and CpG ODN enhanced IL-1β and Th1/Th2 cytokines expression, though with less pronounced effects on IFN-β and NO production. In vivo studies demonstrated that co-administration of Pam3CSK4 with either LPS or CpG ODN synergistically enhanced the Th1/Th2 cytokines expression and iNOS gene expression in the spleen, along with a significant increase in IL-1β and MHC-II expression. Notably, the combination of Pam3CSK4 with LPS also led to a significant induction of IFN-β expression in the spleen and increased nitric oxide (NO) production in the serum, effects that were not observed with the Pam3CSK4 and CpG ODN combination. Overall, our findings highlight the potent synergy between these TLR agonists’ combinations in enhancing Th1/Th2 cytokines responses in chicken, underscoring their potential as effective vaccine adjuvants or immunotherapeutic agents for poultry diseases. Further studies are warranted to elucidate the underlying mechanisms driving this synergistic effect for development of more targeted and effective immune interventions in poultry.

Resiquimod (R-848), a synthetic TLR7 agonist, modulates immune responses, primarily inducing Th1-biased immunity in mammals. In contrast, our previous studies revealed that R-848 stimulates both Th1 and Th2 responses in chickens. The current research investigates the molecular mechanisms underlying these immune responses in chickens. Pooled splenocytes harvested from chickens (n = 2/group) at 24 h post R-848 treatment were subjected to RNA sequencing and significant differentially expressed genes (DEGs) were identified compared to controls. Eight key genes associated with signal transduction (MAPK14, MAP3K8, PIK3CD, STAT1, STAT3, MAPK11, LRRK2, and GATA3) were validated via real-time PCR in chicken peripheral blood mononuclear cells (PBMCs) from six biological replicates. Pharmacological inhibitors or chloroquine were employed to elucidate the signalling pathways. SB202190, IC-87114, and fludarabine phosphate suppressed the R-848induced expression of MAPK14, PIK3CD, and STAT1, respectively, while chloroquine decreased STAT3 expression. Intriguingly, chloroquine treatment enhanced the R-848-mediated expression of MAPK11, LRRK2, and GATA3. These results align with transcriptomic findings and highlight the upregulation of STAT1 and STAT3 as potential contributors to the induction of Th1 and Th2 immune responses by R-848 in chickens. These insights provide a foundation for optimizing R-848 as an immunomodulatory agent in avian species.

Porcine Circovirus 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) of swine and is one of the reasons for severe economic loss in swine industry. In India, there is a considerable prevalence rate of PCV2 infection in pig population, PCV2d being the most prominent genotype. Proper sero-diagnosis and sero-surveillance of the disease is formulated as an effective control measure. In this study, a recombinant capsid protein-based single serum dilution indirect ELISA was developed for determination of antibody titre of the infected pigs. The capsid protein (Cap) of PCV2d was produced in Saccharomyces cerevisiae cells and the capsid protein was purified by affinity chromatography. This recombinant protein was used as a coating antigen to develop a cost effective, highly sensitive and specific single serum dilution ELISA. The in-house developed ELISA was optimized to be used in a 1:200 single serum dilution. The developed ELISA along with a commercial ELISA kit were compared with a sensitive immuno-peroxidase assay (IPMA) by receiver-operating characteristics (ROC) test. Our results showed that the developed single serum dilution ELISA had a higher sensitivity and specificity in comparison to the commercial ELISA. The area under the ROC curve (AUC) also confirmed that the developed ELISA had a better overall diagnostic performance in comparison to the commercial ELISA kit.

Background/Objectives: Porcine Circovirus 2 (PCV2) infection poses significant health and economic challenges to the global swine industry. The disease in pigs leads to lymphoid depletion, resulting in immunosuppression and increased susceptibility to co-infections with other bacterial and viral pathogens. This study evaluated the efficacy of two novel recombinant Newcastle disease virus (NDV) strain R2B vectored vaccines that express the cap gene of PCV2 alone and along with the transmembrane and cytoplasmic tail (TMCT) domains of the NDV F gene. The efficacy of the vaccine candidates was studied in mouse and pig models. Methods: Six-week-old BALB/c mice were divided into five groups and immunized intramuscularly three times at 14-day intervals with various vaccine candidates, namely rNDV-R2B-PCVcap-TMCT, rNDV-R2B-PCVcap, and CircoFLEX commercial vaccine, along with controls. Following immunization and PCV2d virus challenge, multiple assays assessed the immune responses in animal trials. In the pig animal trial, pigs were divided into four groups: a control group (PBS), NDV-vectored PCVcap-TMCT group, NDV-vectored-PCVcap group, and CircoFLEX vaccine group. Pigs were immunized intramuscularly twice at 28-day intervals. Blood samples were collected at regular intervals over 70 days to evaluate the humoral and cell-mediated immune responses. Results: Both mice and pigs’ trials indicated that the NDV-vectored PCV2 cap-TMCT vaccine candidate elicited superior immune responses. In mice, the rNDV-R2B-PCVcap-TMCT group showed enhanced humoral and cellular immunity, increased PCV2-specific antibody levels, higher CD4+/CD8+ ratio, elevated IFN-γ and TNF-α levels, decreased IL-10 levels, reduced viral loads, and minimal histopathological changes. In pigs, the NDV-vectored PCVcap-TMCT group demonstrated better antibody responses, cytokine profiles (IFN-γ and IL-10), and higher levels of PCV2-specific neutralizing antibodies against the PCV2a, PCV2b and PCV2d genotypes when compared to other groups. Conclusions: These findings suggest NDV-vectored PCVcap-TMCT vaccine candidate, expressing the cap gene of PCV2 along with the TMCT domain, offers a promising alternative for protecting against PCV2 infection, potentially addressing the challenges posed by emerging PCV2 strains in the swine industry.

Chicken toll-like receptor 7 (chTLR7) is a viral sensing pattern recognition receptor and detects ssRNA. The ligand binding site comprises leucine-rich repeats (LRRs) located in the ectodomain of chTLR7. Hence, any polymorphism in the binding site would modify its functional interaction with the ligand, resulting in varied strength of immune response. This study first aimed to compare the single nucleotide polymorphisms (SNPs) associated with the ligand binding site of TLR7 in three indigenous chicken breeds namely Aseel, Kadaknath, Nicobari along with an exotic breed White Leghorn. Four synonymous SNPs (P123P, I171I, N339N and L421L) and four non-synonymous SNPs (I121V, S135T, F356S and S447G) were identified among various breeds. We employed in silico tools to screen the pathogenic nsSNPs and one nsSNP was identified as having potential impact on chTLR7 protein. Moreover, sequence and structure-based methods were used to determine the effect of nsSNPs on protein stability. It revealed I121V, F356S, and S447G as decreasing the stability while S135T increasing the stability of chTLR7. Additionally, docking analysis confirmed that I121V and F356S reduced the binding affinity of ligands (R-848 and polyU) to chTLR7 protein. The results suggest that the nsSNPs found in this study could alter the ligand binding of chTLR7 and modify the immune response between different breeds further contributing to disease susceptibility or resistance. Further, in vitro and in vivo studies are needed to analyze the effect of these SNPs on susceptibility or resistance against various viral diseases in poultry.

Infectious bronchitis (IB) is an acute contagious disease of poultry caused by infectious bronchitis virus (IBV). This study investigated the immunopathogenesis and tissue tropism of an Indian IBV field isolate (IBV/Chicken/India/IVRI/Rajasthan/01/2023) in experimental broiler chickens. This isolate belongs to the G1-1 lineage and is closely associated with the Mass genotype. 106.23 EID50/0.2 mL of the virus was administered intranasally and intraocularly to the IBV-challenge group, whereas uninoculated allantoic fluid was administered to the control group. Clinical signs, gross and histopathological lesions, immunohistochemistry (IHC), viral load, humoral responses, and the relative expression of immune response genes were evaluated at seven observation points. The infected group showed a significant reduction in weight gain from 3 dpi onwards, with clinical signs of varying severity from 3 to − 11 dpi. Gross lesions and microscopic changes were observed in the nasal turbinates, trachea, lungs, and kidneys, mainly representing epithelial degeneration and necrosis with mononuclear infiltrates. The caecal tonsils also showed microscopic lesions at 7–9 dpi. Absolute viral load estimation in the organs corroborated the lesion severity scores and IHC results. The expression of innate immune responses broadly demonstrated higher expression in the trachea and lungs of the IBV-infected group during the early phase of infection, whereas similar responses were observed in the kidneys and caecal tonsils during the later phases of infection. This study suggests that the given IBV isolate may cause significant production losses in broilers and exhibit tissue tropism for both respiratory and non-respiratory tissues, triggering varying innate and adaptive immune responses.

PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVADs) which affect pigs worldwide. Currently, there is a worldwide genotype prevalence switch from PCV2b to PCV2d, which has led to increased virulence of the circulating virus strains leading to vaccine failures and selection pressure. In the present study, the PCV2 genotypes circulating in north eastern region (NER) of India particularly the states of Assam and Arunachal Pradesh was characterized by isolation, sequencing and phylogenetic analysis of cap gene. The phylogenetic analysis revealed that the PCV2 isolates circulating in pigs of Assam and Arunachal Pradesh were mostly of PCV2d genotype. Hence, it can be concluded that PCV2d genotype is the most dominating genotype in NER and priority should be given to this genotype for development of future vaccine candidate against PCV2 in India.

Bacterial septicemia causes huge economic losses in the poultry industry and there is no systematic research available in India on the connection of various pathogens associated with septicemia. The present molecular epidemiological study was conducted to investigate the association of different bacterial and immunosuppressive viral pathogens in septicemia suspected chickens. A total of 443 chicken carcasses with septicemic conditions from 71 different flocks were included in this study. Heart blood swabs were subjected to bacterial culture for Salmonella spp., Pasteurella multocida, Escherichia coli, and Gallibacterium anatis. Of these 51 flocks tested for E. coli, 49 (96.1%) flocks were found positive. Among flocks tested for Salmonella spp., 2 flocks were found positive. All tested flocks were found negative for G. anatis and P. multocida as well as air sac swabs tested negative for Mycoplasma spp. Bacterial cultural examination revealed that majority of septicemic chickens were found to be infected with E. coli and these E. coli isolates showed the highest resistance to vancomycin (60%), followed by erythromycin (50%) and cefotaxime (38%) and maximum sensitivity to cefotaxime and clavulanic acid combinations (81.5%), followed by chloramphenicol (69.6%) and ertapenem (67.2%). Among the 5 avian pathogenic E. coli (APEC) virulence genes were detected in 36 flocks and highest frequency of iss (100%), followed by ompT or iutA (97.2%), hly (61.1%) and iroN (47.2%) genes. On polymerase chain reaction (PCR) screening, 10.5, 4.5, 52.2, 19.4, 9.0, 4.5, 20.1 and 19.4% of the flocks were positive for G. anatis, Ornithobacterium rhinotracheale, APEC, Salmonella spp., Mycoplasma gallisepticum, Mycoplasma synoviae, chicken infectious anemia virus and Marek's disease virus, respectively. To our knowledge, the present study is first on the etiology of septicemia in chicken flocks in India. The present study infers that the majority of septicemic deaths in broiler chickens less than 8 weeks have been connected with APEC and majority of E. coli isolates are multidrug resistance, suggesting the need for surveillance and intervention to curb the inadvertent use of antibiotics. Although, incidence of G. anatis association with septicemia was reported, still requires a rigorous epidemiological study to determine the actual prevalence. However, more detailed studies encompassing vast geographical area with large sample size and long duration of the studies are necessary to provide a clear picture of the interaction of different pathogens causing septicemia in chicken.

Various toll-like receptor (TLR) agonists have shown potential as adjuvants with different vaccines in both human and livestock species, including chickens. Our previous studies on combination of lipopolysaccharide (LPS; TLR4 agonist) and resiquimod (R-848; TLR7 agonist) showed the synergistic up-regulation of pro-inflammatory Th1 and Th2 cytokines in chicken peripheral blood mononuclear cells (PMBCs). Hence, the present study aimed to explore the combined adjuvant effect of LPS and R-848 with inactivated Newcastle disease virus (NDV) vaccine in chickens. Two weeks-old SPF chickens were immunized with inactivated NDV vaccine along with a combination of LPS and R-848 as an adjuvant with suitable control groups. A booster dose was given two weeks later. Antibody responses were assessed by enzyme linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test, while cell-mediated immune responses were analyzed by a lymphocyte transformation test (LTT) and flow cytometry following vaccination. Two weeks post-booster, the birds were challenged with a velogenic strain of NDV, and protection against clinical signs, mortality and virus shedding was analyzed. The results indicated that inactivated NDV vaccine with R-848 induced significantly higher humoral and cellular immune responses with 100% protection against mortality and viral shedding following a virulent NDV challenge. However, the combination of LPS and R-848 along with inactivated NDV vaccine produced poor humoral and cellular immune responses and could not afford protection against challenge infection and virus shedding when compared to the vaccine-alone group, indicating the deleterious effects of the combination on antigen-specific immune responses. In conclusion, the combination of LPS and R-848 showed the inhibitory effects on antigen-specific humoral, cellular and protective immune responses when used as an adjuvant with inactivated NDV vaccines in chickens. This inhibitory effect might have occurred due to systemic cytokine storm. A nanoparticle-based delivery of the combination of LPS and R-848 for slow and sustained release could be tried as an alternative method to explore the synergistic effect of the combination as an adjuvant in chickens.

Recombinant Newcastle disease virus vectors have gained a lot of interest for its oncolytic virus therapy and cancer immune therapeutic properties due to its selective replication to high titers in cancer cells. The aim of this study was to find out the oncolytic effects of mesogenic recombinant NDV strain R2B-GFP on murine mammary tumor cell line 4T1 and murine melanoma cell line B16-F10. The anti-tumor effects of R2B-GFP virus were studied via expression of virus transgene GFP in cancer cells, evaluating its cytotoxicity and cell migration efficacies by MTT and wound healing assays respectively. In addition, the underlying apoptotic mechanism of R2B-GFP virus was estimated by TUNEL assay, colorimetric estimation of Caspase-3, 8 and 9 and the estimation of Bax to Bcl-2 ratio. The results showed a significant decrease in viability of both 4T1 and B16-F10 cells infected with R2B-GFP virus at 0.1 and 1 MOI. R2B-GFP virus could significantly induce apoptosis in the 4T1 and B16-F10 cells as compared to the uninfected control. Further, a flow cytometry analysis on apoptotic cells percentage and mitochondria membrane permeability test was also studied in R2B-GFP virus treated 4T1 and B16-F10 cell lines. The R2B-GFP virus caused an increase in loss of mitochondrial membrane permeability in both 4T1 and B16-F10 cells indicating the involvement of mitochondrial regulated cell death. Thus, the recombinant virus R2B-GFP virus proved to be a valid candidate for oncolytic viral therapy in 4T1 and B16-F10 cells.

Chicken anemia virus (CAV) is a non enveloped virus with icosahedral symmetry. It is one of the smallest viruses having diameter of 25 nm that encloses a single stranded circular negative DNA of 2.3 kb size. CAV has been identified as the causative agent of chicken infectious anemia. Chicken infectious anemia disease is also known by name of Blue wing disease, anemia dermatitis syndrome and hemorrhagic aplastic anemia syndrome based on clinical and histopathological lesions. Clinical signs such as anorexia, weakness, stunting growth, unthriftiness, petechiation, ecchymoses, weight loss, anemia can be observed externally in chicks less than 2 weeks of age. However, adult chickens can also gets infected with the virus, though with subclinical symptoms after the maternal antibodies wane. Age related resistance against the disease starts to develop at about 1 week of age and usually gets completed by 2 weeks of age but resistance to infection never develops. Further, CAV once introduced into the flock is considered as a tough virus to deal with because of its resistance to many commonly used disinfectants and its sub-clinical nature in adults and also it is economically devastating to the poultry farmers and hence letting it not into the flock is the only solution. This can easily be achieved by developing highly efficient vaccine against CAV. In view of all the parameters discussed above in this review article we are going to discuss about different types of strategies employed by different research groups for development of high efficacy vaccines against CAV with their advantages and disadvantages. Further we will discuss about what are the future prospect for to increase the efficacy of vaccines.

Newcastle disease virus (NDV) strain R2B, with an altered fusion protein cleavage site, was used as a viral vector to deliver the immunogenic genes VP2 and VP1 of chicken infectious anaemia virus (CIAV) to generate a bivalent vaccine candidate against these diseases in chickens. The immunogenic genes of CIAV were expressed as a single transcriptional unit from the NDV backbone and the two CIA viral proteins were obtained as separate entities using a self-cleaving foot-and-mouth disease virus 2A protease sequence between them. The recombinant virus (rR2B-FPCS-CAV) had similar growth kinetics as that of the parent recombinant virus (rR2B-FPCS) in vitro with similar pathogenicity characteristics. The bivalent vaccine candidate when given in specific pathogen-free chickens as primary and booster doses was able to elicit robust humoral and cell-mediated immune (CMI) responses obtained in a vaccination study that was conducted over a period of 15 weeks. In an NDV and CIAV ELISA trial, there was a significant difference in the titres of antibody between vaccinated and control groups which showed slight reduction in antibody titre by 56 days of age. Hence, a second booster was administered and the antibody titres were maintained until 84 days of age. Similar trends were noticed in CMI response carried out by lymphocyte transformation test, CD4+ and CD8+ response by flow cytometry analysis and response of real time PCR analysis of cytokine genes. Birds were challenged with virulent NDV and CIAV at 84 days and there was significant reduction in the NDV shed on the 2nd and 4th days post challenge in vaccinated birds as compared to unvaccinated controls. Haematological parameters comprising PCV, TLC, PLC and PHC were estimated in birds that were challenged with CIAV that indicated a significant reduction in the blood parameters of controls. Our findings support the development and assessment of a bivalent vaccine candidate against NDV and CIAV in chickens.

Rabies is a devastating disease affecting almost all mammalian animal species including humans. Vaccines are available to combat the disease. Protection against the disease is rendered by assessing the humoral immune response. Recent reports suggest the role of cell mediated immune response (CMI) in assessing vaccine efficacy. In the present study, two live vectored vaccine candidates containing glycoprotein G of rabies virus were generated using the mesogenic Newcastle disease virus (NDV) strain R2B and another with NDV with an altered fusion protein cleavage site as backbones. The efficacy of these vaccine candidates on testing in experimental mouse model indicated generation of robust humoral and CMI responses. The recombinant NDV containing the altered fusion protein cleavage site with glycoprotein G showed the highest CMI response in mice indicating its usage as a potential live vectored vaccine candidate against the disease.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India Abstract: Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of young chickens. Although first observed about 60 years ago, to date, the disease is responsible for major economic losses in the poultry industry worldwide. IBD virus (IBDV), a double-stranded RNAvirus, exists as two serotypes with only serotype 1 causing the disease in young chickens. The virus infects the bursa of Fabricius of particularly the actively dividing and differentiating lymphocytes of the B-cells lineage of immature chickens, resulting in morbidity, mortality, and immunosuppression. Immunosuppression enhances the susceptibility of chickens to other infections and interferes with vaccination against other diseases. Immunization is the most important measure to control IBD; however, rampant usage of live vaccines has resulted in the evolution of new strains. Although the immunosuppression caused by IBDV is more directed toward the B lymphocytes, the protective immunity in birds depends on inducement of both humoral and cell-mediated immune responses. The interference with the inactivated vaccine induced maternally derived antibodies in young chicks has become a hurdle in controlling the disease, thus necessitating the development of newer vaccines with improved efficacy. The present review illustrates the overall dynamics of the virus and the disease, and the recent developments in the field of virus diagnosis and vaccine research.

AbstractLive intermediate plus infectious bursal disease virus (IBDV) vaccines (hot vaccines) are used for protection against the virulent IBDV strains in young chickens. We evaluated the potential of Toll-like receptor (TLR) agonists to alleviate hot vaccine-induced immunosuppression. The combination of Pam3CSK4 and poly I:C synergistically upregulated IFN-β, IFN-γ, IL-12, IL-4, and IL-13 transcripts and cross-inhibited IL-1β, IL-10, and iNOS transcripts in the chicken peripheral blood mononuclear cells (PBMCs) as analyzed by quantitative real-time PCR. Further, four-week old specific pathogen free White Leghorn chickens (n = 60) were randomly divided into six groups and either immunized with hot IBDV vaccine with or without Pam3CSK4 and/or poly I:C or not vaccinated to serve as controls. The results indicated that poly I:C alone and in combination with Pam3CSK4 alleviated vaccine-induced immunosuppression, as evidenced by greater weight gain, increased overall antibody responses to both sheep erythrocytes and live infectious bronchitis virus vaccine, upregulated IFN-γ transcripts and nitric oxide production by PBMCs (P < 0.05), and lower bursal lesion score in the experimental birds. In conclusion, poly I:C alone and its combination with Pam3CSK4 reduced the destruction of B cells as well as bursal damage with restoration of function of T cells and macrophages when used with a hot IBDV vaccine.

Newcastle disease virus (NDV), also known as avian paramyxovirus 1, causes a devastating disease globally in over 250 species of birds known as Newcastle disease. All the viruses belong to a single serotype but categorized into eighteen genotypes based on sequence analysis of the fusion gene. The virus is continuously evolving leading to generation of new genotypes. The clinical manifestation of the disease varies depending on the pathotype of the virus. Very virulent viruses cause severe mortality in susceptible birds whereas less virulent ones cause mild or inapparent symptoms. Diagnosis of the disease is carried out by conventional and molecular tests. The virus can be controlled by live as well as killed vaccines prepared out of less or moderately virulent viruses with considerable level of protection. Recently, genotype-matched vaccines are prepared by reverse genetics for disease control. The virus has a potential to be used as a vector for delivery of foreign immunogenic genes of poultry and other livestock as live-vectored vaccines. Considerable improvements have been made in using the virus as a potential anti-cancer therapeutic for ameliorating cancers of animals and humans. The present chapter delves the various epidemiological dynamics of the virus, diagnosis strategies, and control measures.

Aim: In this study, we have used enzyme-linked immunosorbent assay (ELISA) as an alternative test to replace the cumbersome rapid fluorescent focus inhibition test (RFFIT) to ascertain the immune status of immunized mice against rabies virus. Materials and Methods: Rabies is a devastating disease worldwide caused by rabies virus. Proper usage of pre- or post-exposure rabies vaccine can prevent the disease transmission. In this study, mice were immunized with Vero cell-adapted inactivated rabies vaccine. RFFIT was used as a test to determine the serum neutralizing titers in infected/vaccinated mice. Seroprofiling of mice sera was done in vitro by ELISA. Results: Twenty-one days post-immunization, both ELISA and RFFIT assays indicated similar antibody levels in mice sera that were immunized with Vero cell-adapted inactivated rabies vaccine. Both the tests were correlated, and the linearity was verified by the regression line (R2=0.979). Conclusion: In this study, we profiled the serological status of Vero cell-adapted inactivated rabies vaccine through ELISA in mice model that correlated well with the OIE gold standard test RFFIT.