Genetic grouping of avian infectious bronchitis virus isolated in Brazil based on RT-PCR/RFLP analysis of the S1 gene

Doze isolados de campo do Brasil e uma estirpe de referência vacinal do vírus da bronquite infecciosa das aves (VBI) foram propagadas em ovos embrionados SPF. O gene S1 dessas amostras foi analisado por RT-PCR seguido de RFLP, empregando-se as enzimas de restrição HaeIII, XcmI e BstyI. Observou-se a existência de cinco genotipos diferentes: M (Massachusetts), A , B, C e D. Cinco dos doze isolados de campo do VBI foram classificados no genótipo Massachusetts e os sete vírus restantes foram classificados em quatro genotipos diferentes; A (2), B (2), C (2) ou D (1). Os resultados desta genotipagem concordam com os dados obtidos na análise imunológica previamente realizada para a maior parte destes vírus, destacando a ocorrência de uma variabilidade marcante entre os isolados do VBI que estão circulando nas granjas avícolas comerciais do Brasil.Twelve Brazilian isolates and one reference vaccine strain of avian infectious bronchitis virus (IBV) were propagated in embryonating chicken eggs. The entire S1 glycoprotein gene of these viruses was analysed by reverse-transcriptase-polymerase chain reaction and restriction fragment length polymorphism (RT-PCR-RFLP), using the restriction enzymes HaeIII, XcmI and BstyI. The RFLP patterns led to the classification of these isolates into five distinct genotypes: A, B, C, D and Massachusetts. Five of twelve isolates were grouped in Massachusetts genotype and the remaining seven viruses were classified into four distinct genotypes: A (2), B (2), C (2) or D (1). Such genotyping classification agreed with previous immunological analysis for most of these viruses, highlighting the occurrence of a relevant variability among the IBV strains that are circulating in Brazilian commercial poultry flocks.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Increased expression of Interleukin-6 related to nephritis in chickens challenged with an Avian infectious bronchitis virus variant

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

Molecular and phylogenetic characterization based on the complete genome of a virulent pathotype of Newcastle disease virus isolated in the 1970s in Brazil

Newcastle disease (ND) is caused by the avian paramyxovirus type 1 (APMV-1) or Newcastle disease virus (NDV) that comprises a diverse group of viruses with a single-stranded, negative-sense RNA genome. ND is one of the most important diseases of chickens, because it severely affects poultry production worldwide. In the 1970s, outbreaks of virulent ND were recorded in Brazil, and the strain APMV-1/Chicken/Brazil/SJM/75 (SJM) of NDV was isolated. This strain was characterized as highly pathogenic for chickens but not pathogenic for other bird species. Here we present the complete genome of NDV strain SJM and investigate the phylogenetic relationships of this virus with other NDV strains in terms of genome and proteins composition, as well as characterizing its evolution process. The NDV strain SJM is categorized as a velogenic virus and the complete genome is 15,192 nucleotides in length, consisting of six genes in the order 30-NP-P-M-F-HN-L-5'. The presence of the major pathogenic determinant of NDV strains (R-112-R-Q-K-R down arrow F-117) was identified in the Fusion protein of the NDV strain SJM. In addition, phylogenetic analysis classified the NDV strain SJM as a member of class II, genotype V, and indicates that this virus help us in the understanding of the evolutionary process of strains belonging to this genotype. This study contributes to the growing interest involving the characterization of NDV isolates to improve our current understanding about the epidemiology, surveillance and evolution of the pathogenic strains. (C) 2014 The Authors. Published by Elsevier B. V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Cytokine expression and Mycoplasma hyopneumoniae burden in the development of lung lesions in experimentally inoculated pigs (vol 244, 108647, 2020)

This study aimed to assess immunopathological factors and M. hyopneumoniae (M. hyo) load in macroscopic lesion formation at four timepoints after experimental infection of swine. To do this, 24 M. hyo-free pigs were divided into two groups: non-inoculated control (n = 8) and inoculated (n = 16). At day 0 post-infection (dpi), animals of infected group were intratracheally inoculated with 5 mL of lung inoculum containing 10(7) CCU (Color Changing Units) /mL of M. hyo strain 232, while control group was mock infected with 5 mL of sterilized Friis medium. At 14, 28, 42 and 56 dpi, four animals from the infected group and two from the control group were euthanized and necropsied. The extent of macroscopic lung lobe lesions was visually assessed, scored and lesion samples (qPCR, histopathology and gene expression) were collected. The macroscopic lesion score and estimated M. hyo load (in copies/mu L) at the different timepoints were: 14 dpi: 18.5 %-1.55 x 10(3) copies/mu L; 28dpi: 15.8 %-8.4 x 10(3) copies/mu L; 42 dpi: 7.0 %-3.2 x 10(4) copies/mu L and 56 dpi: 6.3 %-1.11 x 10(5) copies/mu L; Significant and positive correlations between macroscopic lung lesion and the pathogen load were found (coefficient range: 0.77 - 0.99). The cytokine's IL-6 (0.73) and INF-gamma ( - 0.69) gene expression were significantly (p < 0.05) correlated to macroscopic lung lesion score while IL-8, TNF- alpha, IL-1 alpha and IL-1 beta were associated to other pathological effects such as losses in average daily weight gain and microscopic lesion score. The results provide a better understanding about the pathogenicity of M. hyo strain 232 and the host-pathogen interactions, which may be helpful for the development of new treatments or control measures

Cytokine expression and Mycoplasma hyopneumoniae burden in the development of lung lesions in experimentally inoculated pigs

This study aimed to assess immunopathological factors and M. hyopneumoniae (M. hyo) load in macroscopic lesion formation at four timepoints after experimental infection of swine. To do this, 24 M. hyo-free pigs were divided into two groups: non-inoculated control (n = 8) and inoculated (n = 16). At day 0 post-infection (dpi), animals of infected group were intratracheally inoculated with 5 mL of lung inoculum containing 10(7) CCU (Color Changing Units) /mL of M. hyo strain 232, while control group was mock infected with 5 mL of sterilized Friis medium. At 14, 28, 42 and 56 dpi, four animals from the infected group and two from the control group were euthanized and necropsied. The extent of macroscopic lung lobe lesions was visually assessed, scored and lesion samples (qPCR, histopathology and gene expression) were collected. The macroscopic lesion score and estimated M. hyo load (in copies/mu L) at the different timepoints were: 14 dpi: 18.5 %-1.55 x 10(3) copies/mu L; 28dpi: 15.8 %-8.4 x 10(3) copies/mu L; 42 dpi: 7.0 %-3.2 x 10(4) copies/mu L and 56 dpi: 6.3 %-1.11 x 10(5) copies/mu L; Significant and positive correlations between macroscopic lung lesion and the pathogen load were found (coefficient range: 0.77 - 0.99). The cytokine's IL-6 (0.73) and INF-gamma ( - 0.69) gene expression were significantly (p < 0.05) correlated to macroscopic lung lesion score while IL-8, TNF- alpha, IL-1 alpha and IL-1 beta were associated to other pathological effects such as losses in average daily weight gain and microscopic lesion score. The results provide a better understanding about the pathogenicity of M. hyo strain 232 and the host-pathogen interactions, which may be helpful for the development of new treatments or control measures

Dynamics and chronology of Mycoplasma hyopneumoniae strain 232 infection in experimentally inoculated swine

Direct detection of Mycoplasma hyopneumoniae through molecular tools is a growing trend for early diagnosis, highlighting the importance of knowing M. hyopneumoniae dynamics in the respiratory tract upon infection. This study focused on monitoring the infection level and its effects in different anatomic sites of the respiratory tract of experimentally infected swine in four time-points post-infection. To this end, 24 pigs were allocated to either non-inoculated group (n = 8) or inoculated group (n = 16). On day 0 post-infection (dpi), animals of the inoculated group were intratracheally inoculated with M. hyopneumoniae. Nasal swabs were collected weekly for qPCR detection of bacterial shedding. At 14, 28, 42, and 56 dpi, four animals from the inoculated group and two from the control group were necropsied. Bronchoalveolar lavage fluid (BALF) and samples from three different anatomical tracheal sections (cranial - CT, medium - MT, lower - LT) were collected for qPCR and histopathology. Bacterial loads (qPCR) in tracheal samples were: 4.47 x 10(2) copies/mu L (CT), 1.5 x 10(4)- copies/ mu L (MT) and 1.4 x 10(4) copies/mu L (LT samples). M. hyopneumoniae quantification in BALF showed the highest load at 28 dpi (2.0 x 10(6) copies/ mu L). Microscopic lesions in LT samples presented the highest scores at 56 dpi and were significantly correlated with the pathogen load on 14 dpi (0.93) and 28 dpi (0.75). The greatest bacterial load of M. hyopneumoniae in CT samples and BALF was registered at 28 dpi, and it remained high in BALF and LT throughout the 56 dpi. The pathogen was able to persist during the whole experimental period, however higher estimated quantification values were registered in the lower parts of the respiratory tract, especially at 56 dpi. These findings are important for improving diagnostics, treatment, and control measures of M. hyopneumoniae infection in swine herds