Due to the negative impact of Haemonchus contortus in the tropics and subtropics, the detection of serum protein profiles that occur in infected sheep is of high relevance for targeted selective ...treatment strategies (TST). Herein, we integrated proteomics with phenotypic traits to elucidate physiological mechanisms associated to H. contortus infection in susceptible (Dorper – D) and resistant (Santa Inês – S) sheep breeds. Naïve female lambs were infected with H. contortus third-stage larvae on day zero (D0), and samples were collected weekly, for 28 days. Feces were used for individual fecal egg counts (FEC) blood for packed cell volume (PCV) and serum for specific antibody quantification through ELISA. Sera was collected on D0 (-) and D21 (+), and analyzed using a LC-MS/MS based proteomics approach. FEC, PCV, and anti-H. contortus antibody levels confirmed the absence of infection on D0. On D28 there was a significant difference between the two breeds for logFEC means (D = 3774 and S = 3141, p=0.033) and PCV means (D = 16.3 % and S = 24.3 %, p=0.038). From a total of 754 proteins identified, 68 differentially abundant proteins (DAPs) were noted. Phosphopyruvate hydratase (ENO3) was a DAP in all comparisons, while S+ vs D+ and S- vs D- shared the highest number of DAPs (8). Each of the four experimental groups clustered separately in a principal component analysis (PCA) of protein profile. Among the DAPs, proteins associated with the innate and adaptive immune system were detected when comparing S- vs D- and S+ vs D+. In D-, some proteins were linked to stress response to handling, sampling and heat. Focusing on the consequences of infection in each breed, in the D+ vs D- comparison, upregulated proteins were associated with inflammation control and immune response, where downregulated proteins pointed to a negative impact of infection on tissue anabolism, compromising muscle growth and fat deposition. In the S+ vs S- comparison, upregulated proteins were related to immune response, while the downregulated proteins were possibly linked to muscular development and growth, impaired by infection. Collectively, it can be concluded that ENO3 regulation emerges as a potential factor underlying the differential immune response observed between Santa Inês and Dorper sheep infected with H. contortus. In turn, detected acute phase proteins (APPs) reinforce their relation with infection, inflammation and stress conditions, whereas THEMIS-like may contribute to the immune system in Dorper. GSDMD, Guanylate-binding protein and ACAN warrant further investigation as possible biomarkers for TST strategy development.
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•68 out 754 proteins were differentially abundant between Santa Inês and Dorper sheep.•Innate and adaptive immune proteins were associated with response to H. contortus.•Stress-response and muscular-development proteins were impaired by infection.•GSDMD, Guanylate-binding and ACAN are potential biomarkers of H. contortus infection.
Infectious bursal disease (IBD) is an immunosuppressive viral disease of chickens, associated with severe economic losses and major threats to poultry production worldwide. Disease prevention ...programs rely on unequivocal identification of the pathogen, as well as vaccination programs. This study developed a sensitive, one-step, real-time, quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay using a hydrolysis probe system for infectious bursal disease virus (IBDV, VP1 gene) detection and quantification, which was compared to other routinely used diagnostic methods. The assay successfully detected IBD reference viruses and field isolates. The absence of cross-reactivity was detected with negative samples or with other avian viruses in the analytical specificity test. The detection limit of this assay was 70 RNA copies. RT-qPCR was more sensitive in the detection of serially diluted IBDV isolates compared to virus isolation. For clinical samples, the sensitivity and specificity values of RT-qPCR compared to enzyme-linked immunosorbent assay (ELISA) were 97.5% and 100%, respectively, and compared to histopathology, these values were 100% and 93.94%, respectively. RT-qPCR can provide a simple and reliable assay for IBDV surveillance programs and for evaluation of control strategies.
A Brazilian field isolate (IBV/Brazil/PR05) of avian infectious bronchitis virus (IBV), associated with development of nephritis in chickens, was previously genotyped as IBV variant after S1 gene ...sequencing. The aim of this study was to evaluate the levels of IL-6 in kidneys and trachea of birds vaccinated and challenged with IBV/Brazil/PR05 strain, correlating these results with scores of microscopic lesions, specific IBV antigen detection and viral load. The up-regulation of IL-6 and the increased levels of viral load on renal and tracheal samples were significantly correlated with scores of microscopic lesions. Reduced levels of viral load were detected in kidneys of birds previously vaccinated and challenged, compared to non-vaccinated challenged group, although markedly microscopic lesions were observed for both groups. The expression of IL-6, present both in the kidney and in the tracheas, was dependent on the load of the virus present in the tissue, and the development of lesions was related with IL-6 present in the tissues. These data suggest that variant IBV/Brazil/PR05 can induce the expression of proinflammatory cytokines in a manner correlated with viral load and increased IL-6 is involved in the tissue with the influx of inflammatory cells and subsequent nephritis. This may contribute with a model to the development of immunosuppressive agents of IL-6 to prevent acute inflammatory processes against infection with IBV and perhaps other coronaviruses, as well as contribute to the understanding of the immunopathogenesis of IBV nephropatogenic strains.
Uma estirpe variante do vírus da bronquite infecciosa (VBI) associada com o desenvolvimento de nefrite em galinhas, foi isolado e identificado como variante por análise do gene S1. A estirpe IBV/Brazil/PR05 foi testada quanto à sua capacidade de induzir a expressão de interleucina-6 (IL-6) nos tecidos renais e traqueais. Galinhas vacinadas com a estirpe Massachusetts H120 e não vacinadas foram desafiadas com a estirpe IBV/Brazil/PR05. Cinco dias após a infecção, traquéias e rins foram coletados para análise por RT-qPCR, imunohistoquímica e histopatologia. Foi determinada a expressão relativa de IL-6 e da carga viral. A expressão de IL-6 e carga viral foram correlacionadas com o desenvolvimento de nefrite e lesão traqueal. A expressão de IL-6 foi maior quando houve aumento da carga viral na traqueia e nos rins. A carga viral presente nos rins foi inferior quando as aves foram vacinadas, entretanto foi observada nefrite acentuada. Houve alta correlação entre o desenvolvimento de nefrite e o nível de expressão de IL-6, bem como a expressão de IL-6 e a carga viral. A expressão de IL-6, presente tanto nos rins e nas traqueias, foi relacionada a carga viral presente nestes tecidos, e o desenvolvimento das lesões foi relacionado com a expressão de IL-6. Estes dados sugerem que a variante IBV/Brazil/PR05 pode induzir a expressão de citocinas pró-inflamatórias de forma correlacionada com a carga viral, e o aumento de IL-6 está envolvido com o influxo de células inflamatórias no tecido, o que evolui para o desenvolvimento de nefrite. Isto pode contribuir como um modelo para o desenvolvimento de agentes imunossupressores da IL-6 para evitar processos inflamatórios agudos contra infecção com o VBI e talvez outros coronavírus, bem como contribuir para o entendimento da imunopatogênese das estirpes nefropatogênicas deste vírus.
Newcastle disease virus (NDV) causes a fast-spreading, highly contagious infectious disease in several bird species. Commercial poultry farms in Brazil were considered free of virulent NDV. Data on ...NDV infection levels in backyard poultry flocks and the epidemiology of the disease are limited. The aim of this study was to perform a NDV survey in backyard poultry from households flocks located around one of the main wintering sites for migratory wild birds in Brazil, and to identify potential risk factors associated with NDV. Backyard poultry may be sentinels and a source of infection for commercial poultry, since they may have as much contact with these birds as with migratory wild birds. Data were collected from 48 randomly selected households using an epidemiological questionnaire. Serum samples from poultry were tested for NDV antibodies using an ELISA, and tracheal and cloacal swabs were collected for NDV molecular detection. The risk factors were assessed using a multivariate Poisson regression with robust variance. The ELISA showed that 33.8% of the serum samples were positive for anti-NDV antibodies and in 42 households (87.5%) at least one NDV-positive bird was found. Tracheal and cloacal swabs were negative for NDV by real time RT-PCR, possible because within this region there might flow a low pathogenicity NDV strain, which can induce seroconversion with innaparent clinical findings. The prevalence ratio (PR) increased when farmers used their own replacement poultry to restock their flock (PR=1.64; 95% CI: 1.11–2.42). Furthermore, the increasing distance of the household flock from the “Laguna do Peixe” estuary was associated with decreasing NDV seropositivity (PR=0.94; 95% CI: 0.90–0.99). This is the first study in Brazil evaluating the presence of NDV and the associated risk factors in households with backyard poultry flocks. The great number of farms with seropositive birds indicates that the virus circulates in backyard flocks, and this breeding system may be a source of NDV. These data can be used to establish appropriate biosecurity and husbandry measures for this type of breeding system to prevent NDV spread in Brazil.
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