Molecular Characterization of the Chicken Parvovirus Based on VP1 Gene Circulating in Brazilian Chicken Flocks.

Parvovirus infection affects several animal species, especially young animals. In birds, parvovirus infection has been described in Muscovy ducks, turkeys, and chickens, all of which had enteric diseases characterized by diarrhea. Chicken parvovirus (ChPV) has been detected in poultry around the world in animals affected by enteric problems, showing dwarfism, cloacal pasting, and diarrhea. In Brazil, ChPV was detected in chickens affected by diarrhea fifteen years ago. However, the genetic characteristics of ChPV circulating in chicken flocks were not determined. Therefore, the aim of the present investigation was to determine the genetic characteristics of the VP1 gene from ChPV detected in chickens affected by enteric diseases in Brazil. For this purpose, a molecular approach was used. Specific primers were designed to flank the complete VP1 gene of ChPV and amplify it using PCR. The amplified products from samples of chickens with enteric diseases were sequenced, and 22 complete CDs of the VP1 gene were obtained. These samples, compared to the ABU-P1 sequence, showed 17 sequences with high nucleotide (NT) similarity of 92.7-97.4% and amino acid (AA) similarity of 94.8-99.5% associated with Runting and Stunting syndrome (RSS); there were also five samples associated with hens with diarrhea with unusual jejunal dilatation (JD) that had less similarity than the RSS sequences (NT of 86.5% and AA of 93-93.1%). The phylogenetic analysis determined four groups. Group I had sequences from Korea. The second group included sequences from Korea, China, and Brazil (not included in this work). The third group had studied RSS sequences grouped with the ABU-P1 strain and sequences from China and the United States. Finally, the sequences from JD were clustered in a separate group with a bootstrap of 100%, a group that was denoted as group IV, and included sequences from China. RDP4 and SimPlot analysis showed one point of recombination with the sequences of group III ChPV in the JD sequences. Herein, we show that circulating strains of ChPV exhibit genetic differences in the VP1 gene in Brazilian chicken flocks. Nevertheless, more studies are needed to determine the probability of a new genetic group of ChPV based on the analysis of the complete genome.

Development of a fast and sensitive RT-qPCR assay based on SYBR® green for diagnostic and quantification of Avian Nephritis Virus (ANV) in chickens affected with enteric disease.

BACKGROUND: Enteric viruses are among the most prominent etiological agents of Runting-Stunting Syndrome (RSS). The Avian Nephritis Virus (ANV) is an astrovirus associated with enteric diseases in poultry, whose early diagnosis is essential for maintaining a good poultry breeding environment. ANV is an RNA virus that rapidly mutates, except for some conserved regions such as ORF1b. Therefore, the approach of a diagnostic method based on fast-RT-qPCR using SYBR® Green that focuses on the amplification of a fragment of ORF1b is presented as a feasible alternative for the diagnosis of this viral agent. In this study, the proposed assay showed a standard curve with an efficiency of 103.8% and a LoD and LoQ of 1 gene viral copies. The assay was specific to amplify the ORF 1b gene, and no amplification was shown from other viral genomes or in the negative controls. 200 enteric (feces) samples from chickens (broilers) and laying hens with signs of RSS from Ecuadorian poultry flocks were examined to validate the proposed method. RESULTS: Using our method, 164 positive results were obtained out of the total number of samples run, while the presence of viral RNA was detected in samples collected from one day to 44 weeks old in both avian lines. CONCLUSIONS: Our study presents a novel, rapid, robust, and sensitive molecular assay capable of detecting and quantifying even low copy numbers of the ANV in commercial birds, therefore introducing a handy tool in the early diagnosis of ANV in enteric disease outbreaks in poultry.

Comparison of diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 strain vaccines.

Background and Aim: The diagnosis of bovine brucellosis in animals vaccinated with strain-19 (S19) and Rose Bengal (RB)-51 strain vaccines can be misinterpreted due to false positives. This study aimed to compare diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 vaccine strains. Materials and Methods: Two groups of 12 crossbred Holstein calves between 6 and 8 months of age were used. On day 0, blood samples were collected from the animals, and the competitive enzyme-linked immunosorbent assay was used for serological diagnosis of bovine Brucellosis. All animals tested negative. After the first blood collection, the animals were subcutaneously vaccinated: one group received the S19 vaccine and the other received the RB51 vaccine. From the 3rd month after vaccination, all animals were sampled. Sampling was repeated every 2 months until the 7th month. Serological diagnosis of bovine brucellosis was performed using RB, tube serum agglutination test (SAT), SAT with 2-mercaptoethanol (SAT-2Me), and fluorescence polarization assay (FPA). Results: Animals vaccinated with S19 showed positive results with the RB, SAT, and SAT-2Me tests in all months of post-vaccination diagnosis. In animals vaccinated with S19, FPA showed positive results at months 3 and 5 and negative results at month 7, indicating that this test discriminates vaccinated animals from infected animals 7 months after vaccination. Rose Bengal, SAT, SAT-2Me, and FPA tests showed negative results in animals vaccinated with RB51 in all months of diagnosis. Conclusion: Animals vaccinated with S19 may test positive for brucellosis using RB, SAT, or SAT-2Me tests 7 months later. Fluorescence polarization assay is an optimal alternative for diagnosing animals in the field, thereby preventing false positives, and consequently, unnecessary confiscations of animals. Animals vaccinated with RB51 tested negative with RB, SAT, SAT-2Me, and FPA tests in all months of diagnosis, confirming that the tests are ineffective for diagnosing brucellosis caused by rough strains.