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Introduction: Newcastle disease is one of the significant issues in the poultry industry, having catastrophic effects worldwide. The lung is one of the essential organs which harbours Bronchus-associated lymphoid tissue and plays a vital role in the immune response. Leghorn and Fayoumi breeds are known to have differences in resistance to Newcastle disease. Along with genes and long non-coding RNAs (lncRNAs) are also known to regulate various biological pathways through gene regulation. Methods: This study analysed the lung transcriptome data and identified the role of genes and long non-coding RNAs in differential immune resistance. The computational pipeline, FHSpipe, as used in our previous studies on analysis of harderian gland and trachea transcriptome was used to identify genes and lncRNAs. This was followed by differential expression analysis, functional annotation of genes and lncRNAs, identification of transcription factors, microRNAs and finally validation using qRT-PCR. Results and discussion: A total of 8219 novel lncRNAs were identified. Of them, 1263 lncRNAs and 281 genes were differentially expressed. About 66 genes were annotated with either an immune-related GO term or pathway, and 12 were annotated with both. In challenge and breed-based analysis, most of these genes were upregulated in Fayoumi compared to Leghorn, and in timepoint-based analysis, Leghorn challenge chicken showed downregulation between time points. A similar trend was observed in the expression of lncRNAs. Co-expression analysis has revealed several lncRNAs co-expressing with immune genes with a positive correlation. Several genes annotated with non-immune pathways, including metabolism, signal transduction, transport of small molecules, extracellular matrix organization, developmental biology and cellular processes, were also impacted. With this, we can understand that Fayoumi chicken showed upregulated immune genes and positive cis-lncRNAs during both the non-challenged and NDV-challenge conditions, even without viral transcripts in the tissue. This finding shows that these immune-annotated genes and coexpressing cis-lncRNAs play a significant role in Fayoumi being comparatively resistant to NDV compared to Leghorn. Our study affirms and expands upon the outcomes of previous studies and highlights the crucial role of lncRNAs during the immune response to NDV. Conclusion: This analysis clearly shows the differences in the gene expression patterns and lncRNA co-expression with the genes between Leghorn and Fayoumi, indicating that the lncRNAs and co-expressing genes might potentially have a role in differentiating these breeds. We hypothesise that these genes and lncRNAs play a vital role in the higher resistance of Fayoumi to NDV than Leghorn. This study can pave the way for future studies to unravel the biological mechanism behind the regulation of immune-related genes.

Asunto(s)

Pollos , Perfilación de la Expresión Génica , Pulmón , MicroARNs , Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Enfermedades de las Aves de Corral , ARN Largo no Codificante , Transcriptoma , Animales , Pollos/genética , Pollos/inmunología , Enfermedad de Newcastle/inmunología , Enfermedad de Newcastle/virología , Enfermedad de Newcastle/genética , Pulmón/inmunología , Pulmón/virología , Virus de la Enfermedad de Newcastle/inmunología , Virus de la Enfermedad de Newcastle/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/genética , MicroARNs/genética , MicroARNs/metabolismo , Regulación de la Expresión Génica , Resistencia a la Enfermedad/genética , Biología Computacional/métodos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología

RESUMEN

Introduction: Newcastle disease is a highly infectious disease caused by the Newcastle Disease Virus (NDV) and has a devastating financial impact on the global chicken industry. It was previously established that Leghorn and Fayoumi breeds of chicken exhibit variable resistance against NDV infection. The harderian gland is the less studied tissue of the chicken, known to play an essential role in the immune response. Methods: Our previous study, we reported differential gene expression and long noncoding RNAs (lncRNAs) between challenged and non-challenged chickens in the Harderian gland transcriptomic data. Now, we report the analysis of the same data studying the differential expression patterns between Leghorn and Fayoumi and between different timepoints during disease. First, the pipeline FHSpipe was used for identification of lncRNAs, followed by differential expression analysis by edgeR (GLM), functional annotation by OmicsBox, co-expression analysis using WGCNA and finally validation of selected lncRNAs and co-expressing genes using qRT-PCR. Results: Here, we observed that Leghorn showed a higher number of upregulated immune-related genes than Fayoumi in timepoint-based analysis, especially during the initial stages. Surprisingly, Fayoumi, being comparatively resistant, showed little difference between challenged and non-challenged conditions and different time points of the challenge. The breed-based analysis, which compared Leghorn with Fayoumi in both challenged and non-challenged conditions separately, identified several immune-related genes and positive co-expressing cis lncRNAs to be upregulated in Fayoumi when compared to Leghorn in both challenged and non-challenged conditions. Discussion: The current study shows that Leghorn, being comparatively more susceptible to NDV than Fayoumi, showed several immune-related genes and positive co-expressing cis lncRNAs upregulated in challenged Leghorn when compared to non-challenged Leghorn and also in different timepoints during challenge. While, breed-based analysis showed that there were more upregulated immune genes and positive cis-lncRNAs in Fayoumi than Leghorn. This result clearly shows that the differences in the expression of genes annotated with immune-related GO terms and pathways, i.e., immune-related genes and the co-expressing cis-lncRNAs between Leghorn and Fayoumi, and their role in the presence of differences in the resistance of Leghorn and Fayoumi chicken against NDV. Conclusion: These immune-genes and cis-lncRNAs could play a role in Fayoumi being comparatively more resistant to NDV than Leghorn. Our study elucidated the importance of lncRNAs during the host defense against NDV infection, paving the way for future research on the mechanisms governing the genetic improvement of chicken breeds.

RESUMEN

Avian oncogenic or tumor diseases are common in poultry industry causing significant economic loss. Marek's disease (MD), avian leukosis (AL) and Reticuloendotheliosis (RE) are the three major viral oncogenic infections that are difficult to differentiate with gross lesions. Multiplex PCR for simultaneous detection and differentiation of these three viruses was developed and validated. The primers targeting the genes of pp38, pol and LTR for MDV, ALV and REV were designed to yield 206, 429, and 128 bp, respectively. The sensitivity of the PCR primers was checked with serial dilution of positive template DNA for each virus and found to be in the range of 10-5 to 10-7 of 1 µg/µl of initial template DNA. Out of 114 suspected tumor samples screened, 8 samples were positive for MDV, 13 samples were positive for ALV and 31 samples positive for REV. Five samples were positive for both MD and ALV; 3 samples were positive for MD and REV and 25 samples were positive for ALV and REV. Eight samples were positive for all three viruses. Multiplex PCR demonstrated to be a useful technique for simultaneous, rapid detection and differentiation of major tumor causing and immunosuppressive viral diseases of chicken.

Asunto(s)

Enfermedad de Marek , Neoplasias , Enfermedades de las Aves de Corral , Animales , Pollos/genética , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Reacción en Cadena de la Polimerasa Multiplex/métodos , Virus Oncogénicos/genética , Enfermedad de Marek/diagnóstico , Enfermedad de Marek/patología , Enfermedades de las Aves de Corral/diagnóstico , Enfermedades de las Aves de Corral/patología

RESUMEN

The complete genome (2298 nucleotides) of the economically important and immunosuppressive, chicken infectious anemia virus (CAV), from a disease outbreak in a layer flock is discussed. This is the first report of a complete genome sequence of CAV from India. The phylogenetic analyses grouped this isolate with CAV genogroup IIIb based on both complete genome and capsid protein (VP1) sequences. The analyses further revealed the presence of CAV genogroups II, IIIa and IIIb in India. The VP1 sequence identity ranged between 84.4 to 99.3% with that of the Indian isolates and carried a unique substitution at position 447 (serine instead of threonine). Two novel amino acid substitutions were observed at position 52 of VP1 (serine instead of proline) and at position 26 of VP2 (asparagine instead of serine). Sequence analyses of VP1, VP2 and VP3 suggested that the isolate could be attenuated. Comparison with CAV variants, isolated from mammalian species, showed similarities in the numbers of certain transcription factor binding sites in the non-coding regions. Recombination analysis detected no recombination events in this isolate. Further investigations are needed to understand the implications of the unique features of this isolate on viral virulence.

Asunto(s)

Virus de la Anemia del Pollo , Infecciones por Circoviridae , Enfermedades de las Aves de Corral , Animales , Virus de la Anemia del Pollo/genética , Pollos , Infecciones por Circoviridae/epidemiología , Infecciones por Circoviridae/veterinaria , Brotes de Enfermedades , Genotipo , Mamíferos , Filogenia , Serina

RESUMEN

Marek's disease (MD) is a re-emerging viral disease of chickens and a serious economic threat to the poultry industry worldwide. Continuous surveillance with molecular investigation is essential to monitor the emergence of virulent Marek's disease virus (MDV) strains and to devise any appropriate vaccination strategy and implement bio-security programmes. In the present study, we investigated the cases of MD outbreaks in vaccinated poultry flocks. The MD outbreak was confirmed through necropsy (mainly visceral tumours), histopathology and viral gene specific PCR. The pathotypes of the field MDV strains were assessed by molecular analysis of three virulence-associated genes, meq, pp38 and vIL-8. The Meq sequence of the field strains analyzed in this study lacked the 59 aa unique to mild strains, indicating that they are potentially virulent strains. Mutation at position 71 and the presence of five proline rich repeats in the transactivation domain, both associated with virulence were observed in these strains; however, the signature sequences specific to very virulent plus strains were absent. Phylogenetic analysis of meq oncogene sequences revealed clustering of the field strains with North Indian strains and with a very virulent plus ATE 2539 strain from Hungary. Analyses of pp38 protein at positions 107 and 109 and vIL-8 protein at positions 4 and 31 showed signatures of virulence. Sequence and phylogenetic analysis of oncogene and virulence-associated genes of field MDVs from vaccinated flock indicated that these strains possessed molecular features of virulent strains.

Asunto(s)

Herpesvirus Gallináceo 2 , Enfermedad de Marek , Enfermedades de las Aves de Corral , Animales , Pollos , Genotipo , Herpesvirus Gallináceo 2/genética , Enfermedad de Marek/epidemiología , Enfermedad de Marek/prevención & control , Filogenia , Aves de Corral , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/prevención & control , Virulencia/genética

RESUMEN

Oncogenic tumour diseases are major threat to poultry industry. Marek's disease (MD), avian leukosis (ALV) and reticulosendotheliosis virus (REV) are the major tumour causing immunosuppressive viral diseases of chicken. A total of 120 tissue samples presented with tumour lesions from different chicken flocks of coloured broiler, layer breeders and native chicken breeds were screened for MDV, ALV and REV by histopathology and virus specific PCRs individually. Presence of oncogenic viruses in the samples were screened by virus specific PCR. A total of 47 samples were detected either with single infection or dual infection with these viruses. Out of 47, 17 were detected with either one of the viruses and remaining 30 with any of the two viruses. REV was the major cause of tumour in the present samples followed by MDV. ALV was not detected alone, it was either with MD or REV. All 5 ALV positive samples were detected with ALV-E subtype. REV was detected predominantly (22 out of 25 positives) as single infection rather than co-infection.

RESUMEN

Infectious bronchitis virus isolate (IND/AHL/16/01) from a disease outbreak characterized by nephritis, gout and mortality in coloured layer pureline at Directorate of Poultry Research, India was characterized as nephropathogenic strain by S1 genotyping and phylogenetic analysis. Serotyping with homologous and heterologous serum (M41) by virus neutralization assay in embryonated chicken eggs (ECE) showed indices of 7.3 and 2.3 respectively. Pathogenesis, tissue tropism and host immune response induced by this isolate were investigated in experimentally infected chicken. A total of 150, twenty days old seronegative Vanaraja birds were inoculated through intranasal and intravenous route using 104.7 Embryo infective dose50 (EID50/ml). Infected chickens were sacrificed at 4 h, 1, 2, 3, 5, 7, 11, 13, 15- and 20-days post-infection (dpi) for necropsy. Tissues were collected for histopathology and virus detection by isolation in ECE and by reverse transcription- PCR (RT-PCR). Serum was also collected at these intervals to investigate the specific antibody response induced. The symptoms started as early as 3 dpi and included primarily wet droppings, diarrhoea, dehydration rather than respiratory symptoms. Gross lesions were prominent in kidneys including mottling and congestion. Virus isolation and RT-PCR detection indicated the presence of virus as early as 4 h post-infection in trachea and 24 h in kidney and lungs and from 2 dpi in caecal tonsil. The host antibody response after experimental infection in serum by ELISA indicated that the protective titres were induced from 13 dpi and peaked at 35 dpi and declined thereafter. Overall, this isolate is nephropathogenic and capable of inducing severe nephritis and production loss in broilers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00693-4.

RESUMEN

A real time polymerase chain reaction (real-time PCR) assay was developed to detect and quantify the chicken infectious anemia virus (CIAV). The two sets of primers specific to VP1 region of CIAV were designed and their sensitivity and efficacy were studied. Both the primers designed in this study were highly sensitive and were able to detect upto 0.01 fg/µl or 82 × 102 copy number of plasmid DNA. The efficiency of the real time PCR was 100.9%. The results have also shown that the present qPCR assay is 100 times more sensitive than regular qualitative PCR. Both primer sets were validated using 28 field poultry samples and showed good results. The optimized real-time quantitative PCR will be useful in quick detection of field outbreaks, sub-clinical infection in poultry flocks, virus pathogenesis studies and for detecting vaccine contamination.

RESUMEN

Toll-like receptor (TLR) agonists are emerging as promising vaccine adjuvants and immunomodulators in poultry against many diseases. Infectious bursa disease (IBD) still remains as a major threat to poultry industry. Improving the vaccine mediated immune response would help in better protection against IBD virus infection. Adjuvant potential of TLR3 agonist, Polinosinic polycytidylic acid (Poly I:C) with different IBD vaccines has been analyzed in chicken in the present study. Intermediate, intermediate plus IBD vaccine, bursaplex vaccine and their respective poly I:C combinations were used for immunization of chicken. IBD specific antibody titers, bursa to body weight ratio, body weight gain and bursal lesion scores were evaluated at weekly interval in different immunization groups. Fold changes in cytokines IL-1ß and IFN-γ mRNA expression levels in spleen were also analyzed in different groups. Intermediate plus IBD vaccine induced significantly (P ≤ 0.05) higher IBD specific antibody response at 35 days of age than other groups with comparatively lower body weight gain and moderate bursal lesion score. Poly I:C co-administration with intermediate IBD vaccine and bursaplex vaccine improved the IBD specific antibody titers, better body weight gain and moderately less bursal lesion score. However, Poly I:C combination with intermediate plus IBD vaccine did not improve the specific immune response. IL-1ß levels were up-regulated in intermediate plus and bursaplex group, whereas IFN-γ m RNA expression levels were upregulated in intermediate IBD with Poly I:C group. In conclusion, poly I:C co-administration with intermediate IBD and bursaplex vaccine was beneficial and improved the specific immune response with least immunosuppression and bursal damage.

Asunto(s)

Infecciones por Birnaviridae/veterinaria , Pollos , Inmunidad , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Poli I-C/administración & dosificación , Enfermedades de las Aves de Corral/prevención & control , Receptor Toll-Like 3/agonistas , Vacunas Virales/administración & dosificación , Animales , Infecciones por Birnaviridae/prevención & control , Infecciones por Birnaviridae/virología , Enfermedades de las Aves de Corral/virología

RESUMEN

Indigenous chicken breeds are considered to be more disease tolerant than exotic chicken breeds especially for the bacterial diseases. Nicobari and Vanaraja chicken were evaluated for the survivability/mortality patterns and host immune response after experimental infection with P. multocida A1 isolate. The birds were inoculated with 1.9 × 105 CFU/mL through intraperitoneal (I/P) and intranasal (I/N) routes at 2 different age groups viz., 12 wk and 18 wk. Symptoms, mortality rates, lesions in dead birds were observed; Serum from surviving birds of different groups from both breeds were collected at 5, 14, 21, 28, 35 and 42nd d and specific antibody titers were measured by indirect ELISA. At 12 wk of age, the mortality rates were 100% and 16% in birds inoculated by I/P and I/N routes respectively in Vanaraja birds; whereas the mortality rates were 50% and 16% I/P and I/N routes respectively in Nicobari birds. At 18 wk of age the mortality rates were 16% and 50% for I/P routes in Nicobari and Vanaraja birds respectively. The mortality rates were 16% for I/N route in both Nicobari and Vanaraja birds. Lesions such as necrotic foci on liver, congestion in the liver were observed in dead birds. Serum titers were significantly (P < 0.05) higher in surviving Nicobari birds inoculated through I/P route followed by I/N route. The peak titers were reached on 14th d postinfection and declined thereafter. However, no significant difference was found in I/N route of inoculation between 2 breeds. Nicobari chicken breed showed significantly higher survivability and longer mean death time than Vanaraja germplasm to experimental Pasteuralla infection at both the ages however the survivability rate in both breeds improved at later ages.

Asunto(s)

Infecciones por Pasteurella , Pasteurella multocida , Enfermedades de las Aves de Corral , Animales , Pollos , Resistencia a la Enfermedad , Tolerancia Inmunológica , Infecciones por Pasteurella/veterinaria

RESUMEN

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that mediate first line of host defence to pathogens. TLR agonists are potent immunostimulatory agents that help to prime a robust adaptive immune response. In the present study, adjuvant potential of Poly I:C and lipopolysaccharide (LPS) were evaluated with live Newcastle disease virus (NDV) vaccine. Cornish chickens were immunized with live Newcastle disease virus (NDV) vaccine (R2B-mesogenic strain) adjuvanted either with Poly I:C (TLR3 agonist) or LPS-TLR4 agonist and both. Humoral Immune response to ND vaccine was evaluated through haemagglutination inhibition (HI) test and ELISA, while the cellular immune response (CMI) was quantified by lymphocyte transformation test (LTT). IL-1ß cytokine mRNA levels in spleen tissue were also quantified by real time PCR. The results suggest that TLR3 and TLR4 agonists are an efficient immune-stimulators separately, as LPS co-administered group has shown significantly higher serum titre on second week post-immunization and Poly I:C group on third week post-immunization both by HI and ELISA (P < 0.01), however, the combined administration of both LPS and Poly I:C did not give any complementary effect on serum titre. There were no significant differences in stimulation indices (SI) and IL-1ß cytokine levels between groups at different intervals post-immunization. Hence, TLR agonists LPS followed by Poly I:C could be used as adjuvant to enhance the immune response to NDV vaccine in chicken.

Asunto(s)

Inmunidad Humoral/efectos de los fármacos , Lipopolisacáridos/farmacología , Enfermedad de Newcastle/inmunología , Poli I-C/farmacología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 4/agonistas , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Pollos , Ensayo de Inmunoadsorción Enzimática , Inmunidad Celular/efectos de los fármacos , Lipopolisacáridos/administración & dosificación , Virus de la Enfermedad de Newcastle/inmunología , Poli I-C/administración & dosificación

RESUMEN

1. The survivability, innate and adaptive immunity, growth and production traits up to 72 weeks of age were determined in Ghagus, Nicobari (unimproved indigenous) and White Leghorn (WLH) breeds and the study investigated links between innate and adaptive immunity and survivability and production traits.2. At 20 and 40 weeks of age, there was a significant effect of breed on innate immunity assessed by measuring titres of natural antibody (NAb) binding to rabbit red blood cells (RRBC) and adaptive immunity assessed by measuring specific antibody titre (SpAb) to Newcastle disease virus.3. Highest survivability was in WLH (91.6%) followed by Nicobari (87.1%) and Ghagus (82.9%) breeds. Growth traits at different ages were higher (P< 0.001) in Ghagus followed by WLH and Nicobari breeds. Egg production up to 72 weeks was higher (P < 0.001) in WLH followed by Nicobari and Ghagus breeds, whereas egg weight at different ages was higher (P < 0.001) in WLH than Ghagus and Nicobari breeds.4. NAb titres measured at 20 weeks were significantly (P = 0.002) associated with the survivability of hens during 20 to 72 weeks of age. Breed-wise analysis showed a significant (P = 0.019) association between NAb titres at 20 weeks and survivability in the Ghagus breed. Furthermore, NAb titres at 20 weeks were higher in hens which survived to 72 weeks compared with those that died (P = 0.002).5. Measuring NAb titres to RRBC is quick, economical and simple. This method has potential to be used in a breeding programme to increase survivability of laying hens.

Asunto(s)

Pollos/fisiología , Inmunidad Adaptativa , Crianza de Animales Domésticos/métodos , Crianza de Animales Domésticos/normas , Animales , Cruzamiento/normas , Pollos/clasificación , Pollos/crecimiento & desarrollo , Pollos/inmunología , Huevos/normas , Eritrocitos/inmunología , Femenino , Vivienda para Animales/normas , Inmunidad Innata , Modelos Logísticos , Masculino , Conejos

RESUMEN

Birds (364) of both sexes, 11-wk-old, belonging to 2 native (Brown Nicobari and Ghagus) breeds and 1 exotic breed (Dahlem Red) were evaluated for cell-mediated immune response (CMI) by phytohemagglutinin-P (PHA-P), hemagglutination inhibition (HI) assay against Newcastle disease virus (NDV) antigen (LaSota stock virus), flow cytometric analysis of CD8+ cytotoxic T lymphocytes (CTLs), and hematology and biochemical assays. The cutaneous basophil hypersensitivity response PHA-P% increase in wattle thickness (mm) was highest in Ghagus (431.14 ± 22.56) which differed significantly with that of Brown Nicobari (269.1 ± 22.66) and Dahlem Red (218.42 ± 22.30). Sex-wise observation showed that females are having significantly higher response than males. Hemagglutination inhibition test was performed to determine the serum antibodies against Newcastle disease (ND) virus. Brown Nicobari showed highest HI antibody titer than Ghagus and Dahlem Red to similar vaccination program after booster NDV dose. Flow cytometry assay revealed significantly higher CTLs proliferation in Brown Nicobari than Ghagus and Dahlem Red. Moreover, CTLs were found to be higher in control group than the treatment group. Other hematological parameters (103/µL) significant difference was found in white blood cell count between Dahlem Red (38.41 ± 1.03) with that of Brown Nicobari (35.28 ± 1.04) and Ghagus (34.57 ± 1.04) in treatment groups. Same trend was observed in the Lymphocyte treatment group. However, in Granulocyte treatment group, Brown Nicobari (11.04 ± 0.35) was found to be significantly different from Dahlem Red (8.68 ± 0.34) and Ghagus (9.27 ± 0.35). Correlations between body weight at 11 wk of age and CMI, HI, cytotoxic T cell were -0.093, 0.047, and -0.036, respectively. Egg weight was found to be positively correlated with that of chick weight. Serum biochemical values showed that Dahlem Red was having significantly higher creatinine levels compared to Ghagus. Triglycerides level was also significantly higher in Ghagus compared to Dahlem Red. No significant breed effect was observed for alkaline phosphate, aspartate transaminase, and alanine transaminase. Cholesterol and total serum protein levels were significantly higher in Dahlem Red compared to Brown Nicobari.

Asunto(s)

Pollos , Inmunidad Celular , Enfermedad de Newcastle/inmunología , Fitohemaglutininas/antagonistas & inhibidores , Enfermedades de las Aves de Corral/inmunología , Animales , Antígenos Virales/sangre , Linfocitos T CD8-positivos , Femenino , Citometría de Flujo , Pruebas de Inhibición de Hemaglutinación/veterinaria , Masculino , Enfermedad de Newcastle/sangre , Virus de la Enfermedad de Newcastle/fisiología , Enfermedades de las Aves de Corral/sangre

RESUMEN

Pattern recognition receptors (PRR) such as Toll-like receptors, NOD-like receptors, RIG-I helicase receptors, and C-type lectin receptors play a critical role in innate immunity as a first line of defense against invading pathogens through recognition of pathogen and/or damage-associated molecular patterns. Genetic makeup of birds is known to play a role in resistance or susceptibility to various infectious diseases. Therefore, the present study was carried out to elucidate the differential expression of PRR and some of the cytokine genes in peripheral blood mononuclear cells of indigenous chicken breeds such as Ghagus and Nicobari and an exotic chicken breed, White Leghorn (WLH). The stability of expression of reference genes in peripheral blood mononuclear cells of 3 breeds was first determined using NormFinder and BestKeeper programs. NormFinder determined B2M and G6PDH reference genes as the best combination with stability value of 0.38. Out of total 14 genes studied, expression of ten genes was found to be significantly different among 3 breeds after normalization with these reference genes. Ghagus breed showed higher level of expression of TLR1LB, TLR7, NOD1, NOD5, B-Lec, IFNß, IL1ß, and IL8 genes when compared to Nicobari breed. Further, Ghagus showed higher expression of TLR1LB, MDA5, LGP2, B-Lec, IL1ß, and IL8 genes as compared to WLH breed. Higher expression of LGP2 and MDA5 genes was observed in Nicobari compared to the WLH breed while higher expression of TLR7, NOD1, NOD5, and IFNß genes was observed in WLH as compared to Nicobari breed. No difference was observed in the expression of TLR1LA, TLR3, B-NK, and IFNα genes among 3 breeds. Study revealed significant breed effect in expression profile of PRR and some of the cytokine genes and Ghagus breed seems to have better expression profile of these genes linked to the innate immunity when compared to the WLH and Nicobar breeds.

Asunto(s)

Proteínas Aviares/genética , Pollos/genética , Pollos/inmunología , Citocinas/genética , Expresión Génica , Receptores de Reconocimiento de Patrones/genética , Animales , Proteínas Aviares/metabolismo , Citocinas/metabolismo , Perfilación de la Expresión Génica/veterinaria , India , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Receptores de Reconocimiento de Patrones/metabolismo

RESUMEN

Toll-like receptors (TLRs) that sense the microbial pathogens are important components of host immune system. TLRs play key roles in the innate defence mechanism against pathogens, in the development of adaptive immunity, and are possibly the major determinants of the susceptibility to infections. To study the resistance pattern in different breeds of chicken, a comprehensive understanding of TLR4 signalling pathways is required. We investigated the TLR-4 pathway regulated gene expressions in PBMCs of chicken breeds of Broiler (Cobb), Aseel, Dahlem Red and Ghagus upon LPS treatment using Quantitative RT-PCR approach. Several genes were found to be up regulated in both TLR-induced MyD88-dependent and MyD88-independent pathways. These genes include TLR4 (Toll-like receptor 4), MyD88 (Myeloid differentiation primary response gene 88), TRAF6 (TNF receptor associated factor 6), TRIF (TIR domain containing adapter inducing interferon beta), the transcription factors NFkB (Nuclear factor kappa B), IRF7 (Interferon regulatory factor 7) and IFN ß (Interferon beta). We have also studied inflammatory cytokines such as IL2, IL6, IL8, IL1 ß and TNF α to further understand the downstream signalling of TLR4 pathway. These results showed that higher expression of TLR signalling activation via both MyD88-dependent and TRIF-dependent pathways are more beneficial to chicken mononuclear cells mediated innate immunity. We observed TRIF dependent pathway in Aseel and Ghagus breeds. Our results are in concurrent with general observation that Aseel breed is comparatively more resistant, Ghagus and broilers are moderately resistant and Dahlem Red is comparatively more susceptible to bacterial infections.

Asunto(s)

Cruzamiento , Pollos/inmunología , Inmunidad Innata/efectos de los fármacos , Lipopolisacáridos/farmacología , Factor 88 de Diferenciación Mieloide/genética , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Infecciones Bacterianas/inmunología , Citocinas/genética , Resistencia a la Enfermedad/genética , Inmunidad Innata/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.

Asunto(s)

Embrión de Pollo/metabolismo , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Animales , Embrión de Pollo/crecimiento & desarrollo , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Toll-Like/análisis

RESUMEN

Abstract 1. The objective of the experiment was to determine the influence of age, sex and rearing system on Toll-like receptor 7 (TLR7) gene expression in gut, lung and lymphoid tissues and physiological responses to stress in male and female indigenous ducks of Tamil Nadu, India. 2. A total of 36 ducks (12 males and 24 females) were obtained from local farmers and tissue samples of gut tissues (duodenum, jejunum, ileum and caecum), lymphoid organs (spleen and bursa) and lungs were collected in RNAlater solution followed by RNA extraction. 3. After normalisation to ß-actin (endogenous control) qPCR analysis identified a significant effect of age, sex and rearing system on TLR7 expression in the ducks. 4. A significant up-regulation of TLR7 expression was observed in lungs, duodenum, jejunum, ileum and caecum of sexually mature (45 wk) compared with that of immature ducks (16 wk). Among sexes, male ducks had significantly higher TLR7 expression than female ducks. 5. Age and sex interactions were significant in lungs, duodenum, jejunum and caecum. Ducks reared in an extensive housing system showed significantly higher TLR7 expression in bursa, lungs, duodenum, ileum and caecum compared to intensively reared ducks. There were no effects of age, sex and rearing systems on TLR7 expression in the spleen. 6. The heterophil-to-lymphocyte ratio and serum corticosterone were higher in ducks reared on an intensive system compared with ducks from an extensive rearing system.

Asunto(s)

Patos/genética , Tracto Gastrointestinal/metabolismo , Regulación de la Expresión Génica , Vivienda para Animales/normas , Pulmón/metabolismo , Tejido Linfoide/metabolismo , Receptor Toll-Like 7/genética , Factores de Edad , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Patos/metabolismo , Femenino , India , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Factores Sexuales , Receptor Toll-Like 7/metabolismo

RESUMEN

Toll-like receptors (TLRs) are evolutionarily conserved innate immune receptors that recognize pathogen specific molecular pattern (PAMPs) in an efficient, non-self-reactive manner and initiate specific immune signaling that culminates in triggering antigen-specific adaptive responses. Different TLR genes in domestic animal species have been characterized and accumulating evidence from recent studies indicates an extended role for TLR signaling in reproductive physiology. In females, TLRs have been implicated in the regulation of ovulation, fertilization, gestation and parturition, as well as in pathological conditions such as endometritis and mastitis. In males, TLRs play a role in steroidogenesis and spermatogenesis. Use of TLR agonists has also been shown to be effective in the treatment of certain reproductive tract infections. Moreover, gene polymorphisms in TLRs have been associated with mastitis providing evidence that TLRs can potentially be exploited as markers in future breeding programs. The aim of this review is to provide a comprehensive treatise on role of TLRs in male and female reproductive physiology and associated pathology in domestic livestock.

Asunto(s)

Ganado/fisiología , Reproducción/fisiología , Receptores Toll-Like/fisiología , Animales , Femenino , Masculino , Mascotas

RESUMEN

Rabies is an endemic, fatal zoonotic disease in the developing countries. Oral vaccination strategies are suitable for rabies control in developing countries. Studies were performed to investigate the suitability of poly(lactide-co-glycolide) (PLG) microspheres as an oral delivery system for beta-propiolactone inactivated concentrated rabies virus (CRV). Immune responses induced by encapsulated (PLG+CRV) and un-encapsulated inactivated rabies virus after oral and intraperitoneal route administrations were compared. The anti-rabies virus IgG antibody titer, virus neutralizing antibody (VNA) titers obtained by mouse neutralization test (MNT) and IgG2a and IgG1 titers of mice group immunized orally with PLG+CRV showed significantly (p<0.001) higher response than the group immunized orally with un-encapsulated CRV. There was no significant difference (p>0.05) between groups inoculated by intraperitoneal route. The stimulation index (SI) obtained by lymphoproliferation assay of PLG+CRV oral group also showed significantly (p<0.001) higher response than the group immunized orally with un-encapsulated CRV, suggesting that oral immunization activates Th1-mediated cellular immunity. Immunized mice of all experimental groups were challenged intracerebrally with a lethal dose of virulent rabies virus Challenge Virus Standard (CVS). The survival rates of mice immunized orally with PLG+CRV and CRV alone were 75% and 50%, respectively, whereas intraperitoneally immunized groups showed 100% protection. The overall results of humoral, cellular immune response and survival rates of mice immunized orally with PLG+CRV were significantly (p<0.001) higher than those of mice immunized orally with CRV alone. These data suggest that the PLG encapsulated inactivated rabies virus can be used for oral immunization against rabies.

Asunto(s)

Microesferas , Poliglactina 910/farmacología , Vacunas Antirrábicas/inmunología , Rabia/prevención & control , Administración Oral , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Cricetinae , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inyecciones Intraperitoneales , Ratones , Pruebas de Neutralización , Poliglactina 910/administración & dosificación , Propiolactona/farmacología , Vacunas Antirrábicas/administración & dosificación , Distribución Aleatoria , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología