Emerging new avian reovirus variants from cases of enteric disorders and arthritis/tenosynovitis in Brazilian poultry flocks.

1. The objective of this study was to characterise circulating Brazilian avian reovirus (ARV) strains by genetic analysis of the σC protein encoded by segment 1 of the viral genome and compare these with those of viral strains used for immunising commercial poultry.2. The analysis detected the presence of ARV genomes by quantitative reverse transcriptase PCR (RT-qPCR) in the enteric samples and the joint tissues (JT) of birds with signs of viral arthritis/tenosynovitis. Nucleotide sequencing used 16 strains (three commercial vaccines, 10 from enteric tissues and three from JT). The results indicated high variability in the amino acid sequences of 13 wild strains, showing between 40% and 75% similarity compared with the vaccine strains (S1133 and 2177).3. The sequences were grouped into three well-defined clusters in a phylogenetic tree, two of these clusters together with previous Brazilian σC ARV sequences, and one cluster (VII) that was novel for Brazilian strains. Antigenic analysis showed that there were amino acids within putative epitopes located on the surface of the receptor-binding region of the σC protein with a high degree of variability.4. The study confirmed the presence of ARV genetic variants circulating in commercial birds in Brazil, and according to the antigenic prediction, the possibility of antigenic variants appears to be high.

Molecular detection of chicken parvovirus in broilers with enteric disorders presenting curving of duodenal loop, pancreatic atrophy, and mesenteritis.

Enteric disorders are an important cause of economic losses in broiler chickens worldwide. Several agents have been associated with enteric problems, such as viruses, bacteria, and parasites. In this study, broiler chickens showing signs of enteric disorders were subjected to molecular diagnosis for several viral agents and also for pathological examination for elucidating this problem. Thus, the chickens were screened for avian nephritis virus (ANV), chicken astrovirus (CAstV), avian rotavirus (ArtV), avian reovirus (AReoV), infectious bronchitis virus (IBV), fowl adenovirus group I (FAdV-1), and chicken parvovirus (ChPV). Postmortem examinations revealed a curving of the duodenal loop (J-like appearance) and intestines filled with liquid and gaseous content. Histopathological analysis of the duodenal loop showed pancreatic atrophy, acute mesenteritis, and enteritis. PCR results showed that ChPV was the sole viral agent detected in samples with lesions such as the curved duodenal loop and pancreatic atrophy. Molecular characterization of the nucleotide and deduced amino acid sequences revealed a high similarity with other strains of ChPV from Brazil, Canada, United States, Europe, and Asia. These findings suggest an association between ChPV and the development of enteritis, pancreatitis, and pancreatic atrophy, which may lead to curling of the duodenal loop. Together, these alterations may disrupt the normal functioning of the digestive system, diminishing digestion and the absorption of dietary nutrients and consequently leading to reduced weight gain, flock impairment, dwarfism, and an elevated feed conversion rate.

Isolation and molecular characterisation of chicken parvovirus from Brazilian flocks with enteric disorders.

1. The presence of parvovirus in chickens with enteric disease was investigated in commercial flocks in Brazil. 2. The intestinal contents of chickens exhibiting clinical signs of diarrhoea, weight loss or mortality were examined, and chicken parvovirus (chPV) was identified using a polymerase chain reaction (PCR) assay. The samples were sequenced and inoculated into specific-pathogen-free (SPF) embryonated eggs to isolate the virus. 3. Necropsies showed that the embryos were dwarfish, haemorrhagic and oedematous. The presence of chPV was confirmed by PCR and DNA sequencing. 4. The molecular characterisation of chPV strains circulating in the Brazilian flocks showed that they were genetically related to sequences from North America, Europe and Asia. Phylogenetic analyses clustered the Brazilian chPV sequences with those from Europe (Croatia, Hungary) and Asia (South Korea). 5. This study is the first report of the molecular characterisation of chPV circulating in the commercial flocks in Brazil and indicates high genetic similarity with chPV sequences from around the world.

Overcrowding stress decreases macrophage activity and increases Salmonella Enteritidis invasion in broiler chickens.

Overcrowding stress is a reality in the poultry industry. Chickens exposed to long-term stressful situations present a reduction of welfare and immunosuppression. We designed this experiment to analyse the effects from overcrowding stress of 16 birds/m(2) on performance parameters, serum corticosterone levels, the relative weight of the bursa of Fabricius, plasma IgA and IgG levels, intestinal integrity, macrophage activity and experimental Salmonella Enteritidis invasion. The results of this study indicate that overcrowding stress decreased performance parameters, induced enteritis and decreased macrophage activity and the relative bursa weight in broiler chickens. When the chickens were similarly stressed and infected with Salmonella Enteritidis, there was an increase in feed conversion and a decrease in plasma IgG levels in the stressed and Salmonella-infected birds. We observed moderate enteritis throughout the duodenum of chickens stressed and infected with Salmonella. The overcrowding stress decreased the macrophage phagocytosis intensity and increased Salmonella Enteritidis counts in the livers of birds challenged with the pathogenic bacterium. Overcrowding stress via the hypothalamic-pituitary-adrenal axis that is associated with an increase in corticosterone and enteritis might influence the quality of the intestinal immune barrier and the integrity of the small intestine. This effect allowed pathogenic bacteria to migrate through the intestinal mucosa, resulting in inflammatory infiltration and decreased nutrient absorption. The data strengthen the hypothesis that control of the welfare of chickens and avoidance of stress from overcrowding in poultry production are relevant factors for the maintenance of intestinal integrity, performance and decreased susceptibility to Salmonella infection.

Pathogenicity and molecular characteristics of infectious bronchitis virus (IBV) strains isolated from broilers showing diarrhoea and respiratory disease.

Abstract 1. The possibility that infectious bronchitis virus (IBV) variants isolated from broilers with enteric and respiratory problems have a different tropism and pathological outcome from those IBV strains causing classical respiratory disease was investigated. 2. IBV variants were isolated from broiler flocks with enteric and respiratory problems in two regions of Brazil. The USP-10 isolate, of enteric origin, was inoculated via the oral oroculonasal routes into IBV-antibody-free broilers and specific pathogen-free (SPF) chickens to determine tissue tropism and pathogenicity and compared with an IBV variant (USP-50) isolated from chickens showing signs of respiratory disease only. 3. Both USP-10 and USP-50 strains caused similar pathological patterns by either route of inoculation. Both variants were detected in respiratory and non-respiratory tissues, including the kidney, intestine and testis. 4. Broilers were more susceptible to infection than SPF chickens, and seroconversion was detected in all of the chicks.

Enteric viruses in Brazilian turkey flocks: single and multiple virus infection frequency according to age and clinical signs of intestinal disease.

Poult enteritis complex has been associated with enteritis and reduction in growth rates in commercial turkeys worldwide. Intestinal samples from 76 turkey flocks from different Brazilian states affected or not with intestinal disorders were evaluated for the presence of adenovirus groups 1 and 2 (TAV), astrovirus types 1 and 2 (TAstV-1 and TAstV-2), turkey coronavirus (TCoV), reovirus, rotavirus, and avian nephritis virus (ANV) using PCR. The percentage of positive samples was categorized according to the geographic origin, age of the flocks, and presence of clinical signs of intestinal disease. The percentage of samples that were positive for at least one virus was 93.4%, whereas the percentage of samples that were positive for more than one virus was 69.7%. An average of 3.20 viruses per sample was detected in turkeys in the growing phase of the production cycle (1 to 4 wk of age). The TAstV-1 and TCoV were the most frequently observed viruses in growing phase turkeys and occurred simultaneously in 85% of these samples. In turkeys in the finishing phase of development (5 to 18 wk), a lower average number of viruses was observed (2.41), and the most frequent viruses isolated in these turkeys were TAstV-1 (57.1%) and rotavirus (51.8%). Overall, every virus was detected more frequently in growing phase turkeys than in finishing phase turkeys with the exception of TAV. Samples from flocks exhibiting clinical signs of intestinal disease showed a higher rate of positivity, and TAstV-1, TAstV-2, and TCoV were the most frequently occurring viruses in this cohort. Birds without clinical signs most frequently harbored TAstV-1 and rotavirus. Future studies should focus on the description and elucidation of the role of each virus, as well as the pathogenic and immunological implications of the different combinations of viruses in turkeys.

Heat stress impairs performance and induces intestinal inflammation in broiler chickens infected with Salmonella Enteritidis.

Stressful situations reduce the welfare, production indices and immune status of chickens. Salmonella spp. are a major zoonotic pathogens that annually cause over 1 billion infections worldwide. We therefore designed the current experiment to analyse the effects of 31±1°C heat stress (HS) (from 35 to 41 days) on performance parameters, Salmonella invasion and small intestine integrity in broiler chickens infected with Salmonella Enteritidis. We observed that HS decreased body weight gain and feed intake. However, feed conversion was only increased when HS was combined with Salmonella Enteritidis infection. In addition, we observed an increase in serum corticosterone levels in all of the birds that were subjected to HS, showing a hypothalamus-pituitary-adrenal axis activation. Furthermore, mild acute multifocal lymphoplasmacytic enteritis, characterized by foci of heterophil infiltration in the duodenum, jejunum and ileum, was observed in the HS group. In contrast, similar but more evident enteritis was noted in the heat-stressed and Salmonella-infected group. In this group, moderate enteritis was observed in all parts of the small intestine. Lastly, we observed an increase in Salmonella counts in the spleens of the stressed and Salmonella-infected chickens. The combination of HS and Salmonella Enteritidis infection may therefore disrupt the intestinal barrier, which would allow pathogenic bacteria to migrate through the intestinal mucosa to the spleen and generate an inflammatory infiltrate in the gut, decreasing performance parameters.

Heat stress decreases expression of the cytokines, avian ß-defensins 4 and 6 and Toll-like receptor 2 in broiler chickens infected with Salmonella Enteritidis.

A high ambient temperature is a highly relevant stressor in poultry production. Heat stress (HS) has been reported to reduce animal welfare, performance indices and increase Salmonella susceptibility. Salmonella spp. are major zoonotic pathogen that cause over 1 billion of human infections worldwide annually. Therefore, the current study was designed to analyze the effect of heat stress on Salmonella infection in chickens through modulation of the immune responses. Salmonella Enteritidis was inoculated via gavage at one day of age (106cfu/mL). Heat stress 31±1°C was applied from 35 to 41 days of age. Broiler chickens were divided into the following groups of 12 chickens: control (C); heat stress (HS31°C); S. Enteritidis positive control (PC); and S. Enteritidis+heat stress (PHS31°C). We observed that heat stress increased corticosterone serum levels. Concomitantly heat stress decreased (1) the IgA and IFN-γ plasmatic levels; (2) the mRNA expression of IL-6, IL-12 in spleen and IL-1ß, IL-10, TGF-ß in cecal tonsils; (3) the mRNA expression of AvBD4 and AvBD6 in cecal tonsils; and (4) the mRNA expression of TLR2 in spleen and cecal tonsils of chickens infected with S. Enteritidis (PHS31°C group). Heat stress also increased Salmonella colonization in the crop and caecum as well as Salmonella invasion to the spleen, liver and bone marrow, showing a deficiency in the control of S. Enteritidis induced infection. Together, the present data suggested that heat stress activated hypothalamus-pituitary-adrenal (HPA) axis, as observed by the increase in the corticosterone levels, which in turn presumably decreases the immune system activity, leading to an impairment of the intestinal mucosal barrier and increasing chicken susceptibility to the invasion of different organs by S. Enteritidis .

Detection and molecular characterization of chicken astrovirus associated with chicks that have an unusual condition known as "white chicks" in Brazil.

Chicken astrovirus (CAstV) is one of many viruses related to enteric diseases in poultry that are associated with Runting-Stunting Syndrome (RSS), which affects young chickens. CAstV was also recently associated with an unusual condition in chicks called "white chicks." Some hatcheries in certain states of Brazil have reported several incubation problems, mortality, and the presence of chicks with white plumages over the past several months. These chicks were termed locally as "white chicks." The present work investigated 30 chicks with this unusual condition using a multidisciplinary approach. Postmortem examination of each chick showed enlarged livers and intestines that were full of liquid and gas (30/30). The pancreas, kidneys, and spleen were pale (30/30). The other organs did not show any macroscopic alterations. CAstV, chicken parvovirus (ChPV), avian nephritis virus (ANV), avian rotavirus (ARtV), avian reovirus (AReoV), infectious bronchitis virus (IBV), and fowl adenovirus group I (FAdV-1) were tested in the intestines, pancreas, proventriculus, gizzard, liver, spleen, bursa, kidneys, thymus, lung, heart, brain, and yolk sac in each chick. All organs and yolk sacs were positive for CAstV in different titres and negative for the other tested viruses. The partial molecular characterization of the ORF 1b gene of CAstV using 28 sequences revealed a high similarity of the nucleotides and amino acids with sequences of CAstV from North America, Europe, and Asia, and our CAstV sequences clustered into a unique group that was separate from the other sequences. These results demonstrated that CAstV was associated with the white chick condition in Brazil. The virus was distributed in most organs, including the brain and yolk sac. These results suggest that the virus could be transmitted vertically. The molecular characterization also revealed that the CAstV associated with white chick condition was molecularly related to other CAstV sequences found worldwide.

Isolation and molecular identification of lactic acid bacteria and Bifidobacterium spp. from faeces of the blue-fronted Amazon parrot in Brazil.

In Brazil, the blue-fronted Amazon parrot (Amazona aestiva) is a common pet. The faecal microbiota of these birds include a wide variety of bacterial species, the majority of which belong to the Gram-positive lactic acid bacteria (LAB) clade. The aim of this study was to investigate differences in the diversity and abundance of LAB and Bifidobacterium spp. in the cloacae between wild and captive birds and to select, identify and characterise LAB for consideration as a parrot probiotic. Cloacal swabs were collected from 26 wild and 26 captive birds. Bacterial DNA was extracted, and the 16S rRNA genes were amplified. The numbers of PCR-positive Enterococcus, Pediococcus, and Lactobacillus species isolated from wild and captive birds were significantly different (P<0.05). Enterococcus was the most frequently isolated genus, followed by Pediococcus, Lactobacillus, Lactococcus and Bifidobacterium. Enterococcus faecium, Pediococcus pentosaceus, Lactococcus lactis, Lactobacillus coryniformis, Lactobacillus sanfranciscensis and Bifidobacterium bifidum were the most frequently isolated species from all birds. This study increases our understanding of the faecal microbiota, and may help to improve the nutrition and habitat management of captive and wild parrots. The bacterial population identified in the faecal microbiota of clinically healthy wild and captive parrots can serve as a database to analyse variations in the gut microbiota of pathogen-infected parrots and to develop probiotics specific to these genera.

Isolamento e identificação de Bacillus spp de leite UHT: detecção de toxina / Isolation and identification of Bacillus Spp of milk UHT: detection of toxin

O leite produzido a ultra alta temperatura, denominado UHT, é o leite de maior aceitação no mercado consumidor brasileiro. Neste estudo, determinou-se o número de microorganismos mesófilos aeróbios estritos e facultativos viáveis em 65 amostras de leite UHT, procedente de indústrias sob Inspeção Federal no Estado de São Paulo, coletadas durante 2002 e primeiro semestre de 2003. Observou-se que sete amostras (10,76 por cento) estavam fora dos padrões. As produções relativas a duas amostras foram inutilizadas pelo controle de qualidade das empresas, enquanto as demais (7,69 por cento) foram para o mercado consumidor, sendo estas provenientes de duas indústrias processadoras. Analisou-se a presença de Bacillus spp, assim como os eventuais esporos sobreviventes ao processamento, foram isoladas oito cepas de Bacillus spp. Utilizou-se também três (3) cepas de Bacillus spp isoladas pelo Instituto Adolfo Lutz, da cidade de São Paulo, de leite UHT alvo de reclamações de consumidores. Após a identificação morfológica, bioquímica e genética de culturas do Bacillus spp, estudou-se a produção de toxinas por estas amostras, através de inoculação de sobrenadante de cultura estéril em alça ileal ligada de coelho, teste de Dean e em células Vero, Hep2 e fibroblasmo de embrião de galinha. A identificação através de espectroscopio infra-vermelha de Fourier (FT-IR) das amostras isoladas pelo Instituto Adolfo Lutz caracterizou Bacillus flexus, enquanto as amostras isoladas de leite procedente de indústrias apresentaram a presença de B. flexus e B. sporothermodurans (...)