ABSTRACT.-Buim M.R., Mettifogo E., Timenetsky J., Kleven S. & Ferreira A.J.P. 2009 Mycoplasmas are important avian pathogens, which cause respiratory and joint diseases that result in large economic losses in Brazilian and world-wide poultry industry. This investigation regarding the main species of mycoplasmas, Mycoplasma gallisepticum (MG) and M. synoviae (MS), responsible for the above mentioned conditions, was carried out through PCR Multiplex analysis. One thousand and forty-six (1,046) samples of tracheal swabs and piped embryos were collected from 33 farms with laying hens, breeders, broilers or hatchery, located in the Brazilian states of São Paulo, Paraná and Pernambuco, where respiratory problems or drops in egg production had occurred. The MG and MS prevalence on the farms was 72.7%. These results indicated (1) high dissemination of mycoplasmas in the evaluated farms, with predominance of MS, either as single infectious agent or associated with other mycoplasmas in 20 farms (60.6%), and (2) an increase of MS and decrease of MG infection in Brazilian commercial poultry. Epidemiological survey on Mycoplasma gallisepticum and M. synoviae by multiplex PCR in commercial poultry 553

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.

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Many viruses have been associated with runting and stunting syndrome (RSS). These viral infections mainly affect young chickens, causing apathy, depression, ruffled feathers, cloacal pasting, and diarrhea. Chicken Parvovirus (ChPV) is such an infection and has been detected in chickens showing signs of enteric diseases worldwide. Therefore, the present study aims to develop a sensitive real-time fast-qPCR assay based on SYBR® Green for detection and quantification of ChPV. A 561-bp non-structural (NS) gene was amplified and cloned, and a pair of primers was designed based on conserved nucleotide sequences on the NS gene of ChPV, the intercalating DNA reagent SYBR® Green was employed, and the Fast mode of a thermocycler was used. The assay detects 109 to 101 copies of the genome (CG). The limit of detection (LoD) was estimated to five CG, and the limit of quantification (LoQ) was estimated at ten CG. The standard curve efficiency was 101.94%, and the melting curve showed a unique clean peak and a melting temperature of 79.3 °C. The assay was specific to amplify the ChPV NS gene, and no amplification was shown from other viral genomes or in the negative controls. A total of 141 samples were tested using the assay, of which 139 samples were found positive. The highest CG value of ChPV was 5.7 × 106 CG/uL of DNA without apparent clinical signs of enteric disturbance, and 4.6 × 106 CG/uL DNA were detected in chickens with RSS.

Runting-stunting syndrome (RSS) is one of the diseases associated with many detected viruses. In Brazil, there were reports of several enteric disease outbreaks in chickens in which avian nephritis virus (ANV) was detected; however, the role of ANV in the outbreaks and whether the virus was a causative agent of these cases of enteric diseases were not determined. The aim of this study was to isolate ANV in specific pathogen-free (SPF) chicken embryonated eggs (CEE) from the enteric contents of chickens showing signs of RSS. For this purpose, 22 samples of chicken enteric contents that were positive only for ANV were inoculated into 7 and 14-day-old SPF-CEE via the yolk sac route and incubated for 5 d, with a total of 3 passages. Virus isolation was confirmed by the presence of embryo injuries, detection of viral RNA by RT-PCR, and visualization of viral particles using electron microscopy. Therefore, the 7-day-old inoculated embryos showed dwarfism, gelatinous consistency, hemorrhage, and edema in the embryos, whereas the 14-day-old did not show any alteration. Viral RNA was detected in the embryos of both ages of inoculation, and the same viral particles were visualized. The embryos from the mock group showed no alteration and were negative for all the tests. The viral cDNA was sequenced, and the molecular and phylogenetic analyses showed that the Brazilian isolates are more related with the ANV-1 serotype group; the sequences of these isolates showed a high percentage of nucleotide (86.4 to 94.9%) and amino acid (92.3 to 98.7%) similarity with other sequences from China, Japan, Australia, and the United States that belong to this serotype previously classified group. In this study, we isolated 8 samples of ANV in SPF-CEE from enteric content samples from chickens with RSS. In doing so, we showed the pathological injuries to the embryo caused by the virus and the molecular characterization of a part of the ORF 1b gene of the virus.