Complete genome sequence and phylogenetic analysis of feline panleukopenia virus from India.

Parvoviruses are ubiquitous pathogens that cause fatal disease in cats. Feline panleukopenia virus (FPV) is a primitive virus reported first and canine parvovirus (CPV) evolved from FPV and was reported later. Both induce disease in cats and dogs with correlative signs. FPV in domestic cats is genetically diverse and some strains may differ from those used for vaccination. In this study, a virus of FPV strain, ABT/MVC/2022/FPV/001, was identified from a fecal sample of the suspected cat with severe haemorrhagic gastroenteritis. The phylogenetic analysis and complete genome sequence of the strain share 99.75% nucleotide identity with FPV variant MH559110 belonging to Tamil Nadu, India. The results also reveal similarities to strains isolated from Italy, Belgium, and China. The deduced amino acid sequence of isolated strain revealed specific amino acid substitution (Pro5Ala, Phe6Val, His7Gln, Asn9Asp, Lys16Arg, Lys19Arg, Asn52Lys, Gly58Trp, Thr66Ser, Lys67Arg, Leu70His, Asn373Asp and Ala390Thr) which differed from MH559110 and other strains. The complete genomic analysis revealed that the FPV strain circulating in India is evolving rapidly with unique antigenic variations between field FPV, CPV and vaccine strains which may be the major cause for vaccine failure in vaccinated cats. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00854-7.

Molecular detection of porcine parvovirus 1-associated reproductive failure in southern India.

This study used 56 aborted and stillborn fetuses from organized swine farms in Tamil Nadu and Kerala, southern states of India. All samples were screened by using a PCR assay that targets the NS1 gene for PPV. Furthermore, the PCR positive samples were subjected to amplification of the VP2 gene of PPV1 with designed primers and sequenced for further study. The PCR screening of 56 samples found that 14.3% (n = 8) were positive for PPV genome. According to VP2 gene-based PCR for PPV1, 897 bp specific amplicons were detected in all eight of the samples. Two of the eight positive samples (L17 and T5) were sequenced and annotated randomly. The BLAST analysis of contig sequence INDTNCHN-T5 revealed 100% sequence homology with Chinese PPV1genome, whereas sequence from INDTNCHN-L17 revealed 99.43% sequence homology with Spain, Chinese, and German. PPV1 sequences and both the sequences INDTNCHN-T5 and INDTNCHN-L17 were submitted to the GenBank under the accession numbers MW822566 and MW822567 respectively. A phylogenetic analysis of the sequences in this study revealed specific grouping along with PPV1 strains in cluster E. Amino acid analysis of both isolated sequences in addition to the reference sequence from PPV1 showed variations in position 215 (I to T) in both the isolates, variation at position 228 (Q to E) in T5 isolate and variations at position 59 (L to M) and 314 (K to E) in L17 isolate. This study represents the first report of PPV1 cluster E in Tamil Nadu, southern India.

Protein-G-based lateral flow assay for rapid serodiagnosis of brucellosis in domesticated animals.

This study was conducted to develop and evaluate protein-G-based lateral flow assay (LFA) for rapid serodiagnosis of brucellosis in various domesticated animal species. The assay diagnostic performance was tested with 144 reference and 356 field sera samples and then compared with other serological assays. Results revealed that LFA showed 89% and 99% sensitivity and specificity, respectively, when compared with competitive ELISA as the gold standard. This study demonstrated LFA alone as a potential serodiagnostic assay for rapid serodiagnosis of brucellosis in various domesticated animal species.

Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing.

Assessment of cell mediated immune response in rabbits immunized with affinity purified 35 kDa midgut antigen of Rhipicephalus haemaphysaloides ticks.

Cell mediated immune response to immunoaffinity chromatography purified midgut antigen of Rhipicephalus haemaphysaloides ticks in rabbits was studied by using lymphocyte transformation test. This test was carried out by using 5-bromo-2'-deoxy-uridine kit method. The blastogenic response of peripheral blood lymphocytes of normal rabbit to different concentrations of antigen and mitogen (Con A) showed that 2 µg of antigen and 2 µg of mitogen gave maximum stimulation index. The antigen specific responsiveness of immunized rabbits with affinity purified 35 kDa midgut antigen was highly significant (P ≤ 0.01) compared to mitogen. The maximum lymphocyte stimulation index (LSI) of 2.47 was observed on 49 day post immunization in immunized group. The lymphocytes separated from control animal cultured in RPMI1640 medium with 2 µg of antigen and 2 µg of mitogen (Con A) were never stimulated and their LSI values were below 2.0.

Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells.

BACKGROUND: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA) cells, which enable quicker isolation of street rabies virus with minimum passages. OBJECTIVE: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. MATERIALS AND METHODS: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. RESULTS: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. CONCLUSION: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

In ovo delivery of Newcastle disease virus conjugated hybrid calcium phosphate nanoparticle and to study the cytokine profile induction.

In this report, the hybrid calcium phosphate (CaP) nanoparticles were synthesized and functionalized with Newcastle disease virus (NDV). These nanoparticles were synthesized by a combination of co-precipitation and polymerization process and functionalized with amino propyl triethoxy silane before coupling to NDV. The 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay of chicken spleen cells incubated with these nanoparticles indicated that, these particles did not exert any significant cytotoxicity. The effects of hybrid CaP nanoparticles on cell cycle were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of spleen cells were not affected by hybrid CaP nanoparticles compared with their control cells. The hybrid CaP nanoparticles were characterized by scanning/transmission electron microscopy (SEM/TEM); Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction patterns (XRD), Raman spectroscopy and energy-dispersive X-ray spectroscopy (EDX). These methods revealed that NDV was successfully conjugated on nanoparticles. The ability of the hybrid CaP nanoparticles to induce different cytokine mRNAs in the spleen cells of 18-day old embryonated chicken eggs (ECEs) was studied by quantitative real time polymerase chain reaction (qRT-PCR). NDV conjugated particles induced a high expression of Th1 cytokines such as interferon (IFN)-α, tumor necrosis factor (TNF)-α of and Th2 cytokines, interleukin (IL) 6 and IL-10. Uncoupled NDV induced only Th1 cytokines, IFN-α, INF-γ and TNF-α. The hybrid particles alone did not induce any cytokines. This confirmed that nanoparticle coupling could induce differential cytokine profiles and hence can be used as an alternate strategy to direct favorable immune responses in animals or chickens using appropriate vaccination carrier.

Rapid determination of hydrogen peroxide produced by Lactobacillus using enzyme coupled rhodamine isocyanide/calcium phosphate nanoparticles.

A sensitive method for detecting hydrogen peroxide (H2O2) using rhodamine isocyanide incorporated calcium phosphate nanoparticles (Rho/CaP) was developed. The synthesized nanoparticles were characterized based on transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR), Raman spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction patterns (XRD). To study the application, the nanoparticles were functionalized with horse radish peroxidase (HRP) based on aminopropyl triethoxy silane (APTES) and used as tools to detect H2O2. The detection strategy was based on fluorescence quenching or colorimetric detection. The enzyme immobilized nanoparticles were titrated with different concentrations of H2O2 and a fixed concentration of O-phenylenediamine (OPD). The HRP conjugated Rho/CaP strongly catalyzed H2O2 oxidation of OPD that caused fluorescence quenching at 575 nm. For colorimetric detection, the OPD product was read at 492 nm. In the fluorescence quenching assay, the minimum detectable concentration was ~1 pmol in contrast to ~5 nmol in the colorimetric assay. The minimum detectable concentration by visual detection was ~500 nmol. The specificity of the developed assay method was examined with different interferences which did not produce any significant response. This assay was applied, along with a commercially available kit to compare the H2O2 production capacities of different Lactobacillus strains. The results indicated that the developed assay and commercially available kit methods were highly correlated. The fluorescence quenching kinetics is also discussed.

Comparative analysis of innate immune response following in vitro stimulation of sheep and goat peripheral blood mononuclear cells with bluetongue virus - serotype 23.

Bluetongue is an infectious disease caused by bluetongue virus (BTV), which affects sheep, goat, cattle and certain wild ruminants. However severe clinical signs are usually seen with significant mortality in sheep than cattle and goat. To date, comparative studies on innate immune responses of sheep and goat infected with BTV is lacking. In this study, we compared the innate immune response of sheep and goat by infecting the peripheral blood mononuclear cells (PBMCs) with BTV serotype 23. In our study, we observed that sheep PBMCs supports higher virus replication than goat PBMCs. To delineate the role of innate immune response in differential viral replication observed in this study, we examined TLR3 (Receptor for dsRNA virus) mRNA expression and cytokine profiles (IL-1ß, Il-6, IL-8, Il-10, IL-12p40, TNF-α, IFN-γ and IFN-α) following Poly I:C (TLR3 ligand) stimulation and BTV 23 infection. In our present study, sheep PBMCs had significantly higher TLR3 mRNA levels, TLR3 specific ligand (Poly I:C) stimulation resulted in increased levels of IFN-γ at transcriptional and translational levels along with IL-8 and IL-10 at transcriptional levels. Whereas, the levels of TNF-α was higher in goat PBMCs at transcriptional levels. BTV infected sheep PBMCs expressed significantly higher levels of IFN-γ at transcriptional and translational levels along with IL-6, IL-8 and IL-10 at transcriptional levels. Whereas the expression levels of TNF-α and IFN-α at transcriptional and translational levels were significantly high in goat PBMCs. To examine the potential factor for consistent increase in the expression of TNF-α, we sequenced the promoter region of TNF-α and identified a total of five single nucleotide polymorphisms (SNP) and one indel in goat TNF-α promoter region. Luciferase assay for transcriptional activity of the promoter showed that goat TNF-α has significantly enhanced transcriptional activity in comparison with sheep TNF-α promoter. Altogether, our data suggests that the expression levels of TNF-α and IFN-α and/or IL-10 plays crucial role in replication of BTV 23.

Real-time PCR-based quantification of Eimeria genomes: a method to outweigh underestimation of genome numbers due to PCR inhibition.

Eimeria species parasites can cause the disease coccidiosis in all livestock species, most notably poultry. Traditional diagnostics such as faecal microscopy have now been supplemented by molecular assays including genus-specific and species-specific quantitative polymerase chain reaction (qPCR), although DNA extracted from faecal samples is commonly affected by PCR inhibition. This was confirmed when genomic DNA extracted from chicken faeces inhibited the threshold cycle value of internal positive control (IPC) DNA amplification by 15.33%. Hence, the objective of the present study was to use IPC qPCR to determine PCR inhibition in a series of experimental samples and use the increase in IPC qPCR threshold cycle value as an individual (sample-specific) correction factor for an established 5S rDNA qPCR used to estimate total Eimeria genome numbers. IPC-corrected genome counts were correlated with conventional oocyst per gram counts and compared with non-corrected counts, revealing a 0.1769 increase in correlation coefficient to outweigh underestimation of oocyst counts. Though the sample size used in this study is small, this limitation would be offset by the sample-specific correction factor determined using the IPC along with each sample.

Foetal stem cell derivation & characterization for osteogenic lineage.

BACKGROUND & OBJECTIVES: Mesencymal stem cells (MSCs) derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. METHODS: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR) analysis and confirmed by sequencing using genetic analyzer. RESULTS: Ovine foetal samples were processed to obtain mononuclear (MNC) cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45⁻/CD14⁻ was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinß1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP) activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. INTERPRETATION & CONCLUSIONS: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established. Criteria proposed for defining MSCs by this study includes the cell adherence to culture plates, specific surface protein profiles and differentiation to osteogenic lineage. The MSCs and osteogenic differentiated cells in this ovine animal model may serve as a large source for stem cell applications in regenerative medical therapies.

Factors influencing on prevalence of Cryptosporidium infection in south Indian dairy calves.

The objectives of the present study were to report the influence of factors like age, sex, breed, dung consistency and rearing system on prevalence of Cryptosporidium spp. in south Indian cattle. Two-step nested PCR was employed for detection of Cryptosporidium infection in dairy calves of south Indian states viz., Andhra Pradesh, Karnataka, Kerala, Tamil Nadu and union territory i.e., Puducherry. A total of 459 dung samples from the calves were subjected to nested PCR, 182 were found positive with prevalence percent of 39.65. Age wise comparison showed a high prevalence of Cryptosporidium in the age group of one month old calves. This concludes that the cryptosporidiosis is highly age dependent with young calves showed the highest prevalence. Depending on the group had consistency of dung, the highest prevalence of Cryptosporidium was observed in semi-solid dung, followed by formed and the diarrhoeic group animals. Female calves showed slightly higher prevalence rate than male animals. Cow calves had an overall prevalence percent of 40.75 and the infection rate in buffalo calves was 36.28 %. In relation to rearing system, individual animals had 42.18 % and farm animals showed 38.46 % of Cryptosporidium infection. In conclusion, the prevalence of Cryptosporidium in dairy calves should be correlated with the factors like age, sex, breed, dung consistency and rearing system of the animal to arrive at a reliable epidemiological data on bovine cryptosporidiosis.

Expression profile of toll-like receptor 2 mRNA in selected tissues of shark (Chiloscyllium sp.).

Sharks are a species of delight for immunologists from the evolutionary perspective since it is considered as the first species to have evolved the adaptive immune responses in addition to the innate immune system. One of the components of the highly conserved innate immune system is the toll-like receptors (TLR) which has a conserved overall protein structure throughout deuterostome evolution. There is no report that demonstrates the expression of these receptors in sharks. In this study we successfully amplified a 270 bp amplicon using a degenerate primer design strategy that corresponded to the Toll/IL-1 receptor (TIR) domain of TLR2 (GenBank ID: JF792813). BLAST analysis revealed a maximum nucleotide identity of 87% and 76% with the TLR2 of higher mammals and teleost fishes respectively. Domain prediction revealed a TIR structure between 1 and 87 amino acids that had a maximum identity of 58% and 76% with TLR2 - TIR protein of teleost fishes and higher mammals respectively. Phylogenetic analysis revealed a closer clustering of the shark TIR sequence with those from human, cattle, goat, sheep and chicken than with other fish species. Basal expression levels of the TLR2-TIR mRNA were found to be significantly higher in kidneys followed by fins, spleen and intestinal spiral valve (ISV). In tissues such as spleen and kidney the expression of the TLR2-TIR mRNA could be localized to lymphoid and macrophages like cells and tubular epithelial cells respectively. In-vivo exposure of sharks to peptidoglycan (TLR 2 ligand) resulted in 9 folds higher expression of TLR2-TIR mRNA in gills followed by 5 folds in the fins. However, when inoculated with a TLR ligand pool, the expression levels significantly increased to 12 fold in skin followed by epigonal, kidneys and ISV. These findings not only support the presence of the TLRs in sharks but also their induction upon exposure to specific ligands. Further studies are needed to identify their numbers, their ligand specificity and downstream cytokine responses.

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India.

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.

Molecular prevalence of Cryptosporidium spp. in dairy calves in Southern states of India.

Dung samples were collected from dairy calves of south Indian states viz., Andhra Pradesh, Karnataka, Kerala, Tamil Nadu and union territory, Puducherry and are subjected to nested polymerase chain reaction (PCR) targeting 18S rRNA gene for detection of Cryptosporidium infection. Of the 459 dung samples screened 182 were found positive with a prevalence of 39.65%. Highest prevalence of Cryptosporidium was observed in Puducherry (86.67%) and lowest in Kerala (17.65%). Genotyping by PCR-restriction fragment length polymorphism (RFLP) and sequence analysis revealed the presence of all the four major Cryptosporidium species of cattle viz., Cryptosporidium andersoni, Cryptosporidium ryanae, Cryptosporidium parvum and Cryptosporidium bovis. C. andersoni was widely distributed in calves of Tamil Nadu, Karnataka and Puducherry whereas in Andhra Pradesh C. ryanae was the major species. Of the 64 samples subjected to PCR-RFLP, 39 (60.94%) could be classified as C. andersoni, 18 (28.13%) as C. ryanae, 4 (6.25%) as C. parvum and 3 (4.69%) were confirmed as C. bovis. The results were also confirmed by sequencing of 19 Cryptosporidium DNA samples.

Cloning and sequencing of Indian water buffalo (Bubalus bubalis) interleukin-3 cDNA.

Full-length cDNA (435 bp) of the interleukin-3(IL-3) gene of the Indian water buffalo was amplified by reverse transcriptase-polymerase chain reaction and sequenced. This sequence had 96% nucleotide identity and 92% amino acid identity with bovine IL-3. There are 10 amino acid substitutions in buffalo compared with that of bovine. The amino acid sequence of buffalo IL-3 also showed very high identity with that of other ruminants, indicating functional cross-reactivity. Structural homology modelling of buffalo IL-3 protein with human IL-3 showed the presence of five helical structures.

Development and evaluation of flow through technique for diagnosis of cystic echinococcosis in sheep.

A novel in vitro flow through technique was developed and evaluated for immunodiagnosis of cystic echinococcosis in sheep using hydatid specific non-cross reactive 8-kDa protein. The 8-kDa protein was prepared from hydatid cyst fluid by DEAE-Sepharose fast flow anion exchange chromatography. In this flow through technique, the 8-kDa antigen was coated on the nitrocellulose membrane of flow through device. Protein A colloidal gold was used as detector. The evaluation of the technique was performed by comparing 150 known positive hydatid serum and known negative serum collected from sheep. The test was shown to be high sensitivity and specificity that were closely correlated with those of EITB. Furthermore the immunofiltration-based assay is rapid (2 min) and easy to perform with no requirement of special skill, reagent and instrumentation. This suggests the flow through technique is an acceptance alternative to be used in clinical laboratories lacking specialized equipments as well as large scale screening of cystic echinococcosis both in the field with animal and human populations.

Sequence analysis of Toll-like receptor genes 1-10 of goat (Capra hircus).

This study involved cloning and sequencing of the coding regions of all 10 Toll-like receptor (TLR) genes of goat. Goat TLR 1-10 gene sequences revealed a high degree of nucleotide identity with sheep and cattle sequences (>90%) and 75-85% with pig, mouse and human sequences. At the amino acid level, 85-99% similarity was observed with sheep and cattle and 60-85% with pig, mouse and human. TLR9c DNA of goat showed the highest amino acid identity to that of sheep (99%) while TLR8 cDNA showed the lowest identity of 88.7% to that of sheep. Variations were seen in the number of leucine rich repeats (LRRs) of goat TLRs as compared to other ruminant species with maximum differences in the TLR3 gene. Phylogenetic analysis through molecular evolution and genetic analysis (MEGA) software and multi dimensional scaling revealed a high degree of conservation of goat TLRs with those from other species. However when the TIR domain of all the TLRs were compared, goat TLR7 TIR alone showed a high divergence of 19.3 as compared to sheep sequences. This is the first report of the full-length cDNA sequences of all the 10 TLR genes of goats which would be a useful tool for the study of evolutionary lineages and for phylogenetic analysis.

Calcium phosphate coupled Newcastle disease vaccine elicits humoral and cell mediated immune responses in chickens.

Calcium phosphate (CaP) particles were coupled with inactivated Newcastle disease virus (NDV) vaccine. The surface morphology of CaP particles coupled to NDV was found to be spherical, smooth and with a tendency to agglomerate. The mean (± SE) size of CaP particles was found 557.44 ± 18.62 nm. The mean percent encapsulation efficiency of CaP particles coupled to NDV assessed based on total protein content and haemagglutination (HA) activity in eluate was found to be 10.72 ± 0.89 and 12.50 ± 2.09, respectively. The humoral and cell mediated immune responses induced by CaP coupled NDV vaccine were assessed in comparison to a commercial live vaccine (RDV 'F'). CaP coupled NDV vaccine elicited prolonged haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titres in the serum even at fourth and fifth week post-vaccination (PV), unlike RDV 'F' inoculated chickens whose titres declined to insignificant levels by this time. CaP coupled NDV vaccine could stimulate HI antibodies in tracheal washings and tears from second and first week PV, respectively. IgA ELISA antibodies were also seen in tracheal washings of these birds from third week PV and in tears from second week PV. CaP coupled NDV vaccine elicited cell mediated immune responses (CMI) from two to four weeks PV. The stimulation indices obtained after stimulation with specific antigen was not significantly different between CaP coupled antigen and live NDV virus except on first week PV. However, CaP coupled antigen did not cause suppression of lympo proliferation as indicated by statistically similar responses to mitogen, concanavalin A between the two groups. Overall, CaP coupled NDV vaccine elicited stronger and prolonged immune responses in comparison to the commercial live vaccine. No increase in the serum calcium and phosphorous levels were seen in CaP coupled NDV vaccine inoculated chickens.

Changes in the Cytokine and Toll-Like Receptor Gene Expression Following Infection of Indigenous and Commercial Chickens With Infectious bursal disease virus.

A comparative study of cytokine and toll-like receptor (TLR) mRNA expression in 3 weeks old indigenous and commercial chickens infected with a very virulent strain of Infectious bursal disease virus (IBDV) was performed using a custom-made microarray chip. In uninfected indigenous chickens, the basal levels of interleukin (IL) 15 were lower and IL 16 was higher than their commercial counterparts. In the IBDV infected indigenous chickens, only IL16 gene expression was down regulated, while TLR3 expression was up regulated significantly. In the IBDV infected commercial chickens IL15, IL16 and TLR3 were down regulated. But, IL1-ß, IL2, IL8, IL12, IL17, interferon (IFN)-α and ß were significantly increased compared with the control. In IBDV infected indigenous chickens, IL15, IFN-γ, beta-defensin and TLR3 were up regulated compared to virus-infected commercial chickens. The results suggested that up regulation of TLR3, a ligand for double-stranded (ds) RNA probably could account for the possible clinical resistance in these birds. There was a 5.2 fold difference by quantitative real-time RT-PCR between indigenous and commercial chickens in TLR3 mRNA expression. Therefore, TLR3, a receptor for dsRNA could be a putative molecule that could play a role in differential innate and adaptive immune responses to IBDV in commercial and indigenous chickens.