Quantitative analysis of waterfowl parvoviruses in geese and Muscovy ducks by real-time polymerase chain reaction: correlation between age, clinical symptoms and DNA copy number of waterfowl parvoviruses

<p>Abstract</p> <p>Background</p> <p>Waterfowl parvoviruses cause serious loss in geese and ducks production. Goose parvovirus (GPV) is infectious for geese and ducks while Muscovy duck parvovirus (MDPV) infects Muscovy ducks only. So far, for these viruses' sensitive detection polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were applied. However, there was no molecular biology method for both waterfowl parvoviruses detection and quantification which could unify the laboratory procedures. The level of GPV and MDPV replication and distribution plays a significant role in the parvoviral infection progress and is strictly correlated to clinical symptoms. Meanwhile, experiments conducted previously on GPV distribution in geese, performed as animal trial, did not involve epidemiological data from the disease field cases. The study on the correlation between age, clinical symptoms and viral DNA copy number may be benefitable in understanding the GPV and MDPV infection. Such data may also aid in determination of the stage and severity of the infection with parvoviruses. Therefore the aim of this study was to develop quantitative real-time PCR for parallel detection of GPV and MDPV in geese and Muscovy ducks and to determine the correlation between the age of the infected birds, clinical symptoms and DNA copy number for the estimation of the disease stage or severity.</p> <p>Results</p> <p>In order to develop quantitative real-time PCR the viral material was collected from 13 farms of geese and 3 farms of Muscovy ducks. The designed primers and <it>Taqman </it>probe for real-time PCR were complementary to GPV and MDPV inverted terminal repeats region. The pITR plasmid was constructed, purified and used to prepare dilutions for standard curve preparation and DNA quantification. The applied method detected both GPV and MDPV in all the examined samples extracted from the heart and liver of the infected birds. The conducted correlation tests have shown relationship between age, clinical symptoms during parvoviral infection and the DNA copy number of these pathogens. The method allowed for a sensitive detection of GPV and MDPV even in 1-week old infected goslings or 2-week old ducklings before observation of any disease symptoms.</p> <p>Conclusions</p> <p>The developed method was found to be a valuable tool for the unification of laboratory procedures and both parvoviruses parallel detection and quantification. The conducted analysis revealed significant correlation between the age of the infected birds, the observed clinical symptoms and DNA copy number of GPV and MDPV in the examined organs. The obtained data may aid in better understanding of the pathogenesis and epidemiology of Derzsy's disease and 3-w disease as well as estimation of the infection's severity and stage of the disease.</p

Marek’s Disease Is a Threat for Large Scale Poultry Production

Marek’s disease (MD) is one of the widespread infectious diseases that causes huge losses in large-scale poultry production. This is due to weight loss, poorer feed conversion and an increased number of deaths among infected birds. The etiological agent is a Marek’s disease virus (MDV) belonging to the Herpesviridae family. It is mainly described in poultry, however, it is also found in geese. There are three MDV serotypes, and four patotypes within serotype 1. Currently, Marek’s disease is very rare in its classical form. There are non-specific clinical symptoms, and anatomopathological changes are mainly observed in the liver, spleen and the reproductive system. This may be due to the evolution in the pathogenicity of MDV field strains over the past several decades. The presence of MDV and number of molecular diagnostic tests based on the detection of viral nucleic acids and viral proteins is already found in birds that have several weeks old. Laboratory diagnostics are based mainly on molecular biology (mainly PCR) methods. The only relatively effective method instead of biosecurity measures, of preventing MD is prophylactic vaccination of 1-day-old chickens or in ovo vaccination. Nevertheless, Marek’s disease is still recorded in poultry flocks around the world, with estimated losses reaching several million dollars

Molecular characteristics of Polish field strains of Marek's disease herpesvirus isolated from vaccinated chickens

<p>Abstract</p> <p>Background</p> <p>Twenty-nine Marek's disease virus (MDV) strains were isolated during a 3 year period (2007-2010) from vaccinated and infected chicken flocks in Poland. These strains had caused severe clinical symptoms and lesions. In spite of proper vaccination with mono- or bivalent vaccines against Marek's disease (MD), the chickens developed symptoms of MD with paralysis.</p> <p>Because of this we decided to investigate possible changes and mutations in the field strains that could potentially increase their virulence. We supposed that such mutations may have been caused by recombination with retroviruses of poultry - especially reticuloendotheliosis virus (REV).</p> <p>Methods</p> <p>In order to detect the possible reasons of recent changes in virulence of MDV strains, polymerase chain reaction (PCR) analyses for <it>meq </it>oncogene and for long-terminal repeat (LTR) region of REV were conducted. The obtained PCR products were sequenced and compared with other MDV and REV strains isolated worldwide and accessible in the GeneBank database.</p> <p>Results</p> <p>Sequencing of the <it>meq </it>oncogene showed a 68 basepair insertion and frame shift within 12 of 24 field strains. Interestingly, the analyses also showed 0.78, 0.8, 0.82, 1.6 kb and other random LTR-REV insertions into the MDV genome in 28 of 29 of strains. These genetic inserts were present after passage in chicken embryo kidney cells suggesting LTR integration into a non-functional region of the MDV genome.</p> <p>Conclusion</p> <p>The results indicate the presence of a recombination between MDV and REV under field conditions in Polish chicken farms. The genetic changes within the MDV genome may influence the virus replication and its features <it>in vivo</it>. However, there is no evidence that <it>meq </it>alteration and REV insertions are related to the strains' virulence.</p

Avian influenza - global epidemic problem

Avian influenza is a contagious disease of animals caused by viruses, members of the family Or-thomyxoviridae, genus influenzavirus A. Influenza A viruses have 16 H subtypes and 9 N subtypes. Only viruses of the H5 and H7 subtypes are known to cause the highly pathogenic form of the disease. Wild waterfowl are considered the natural reservoir of all influenza A viruses. The role of migratory birds in the spread of highly pathogenic avian influenza is not fully understood. They have probably carried influenza viruses and they can introduce to poultry flock H5 and H7 viruses, which can mutate to the highly pathogenic form. Some migratory birds are now directly spreading the H5N1 virus in its highly pathogenic form. The widespread persistence of H5N1 in poultry population is dangerous for human health. In Hong Kong, H5N1 virus infected 18 people and killed 6 of them in 1997 and the second time in early 2003 the virus caused two infections, with one death. The virus has appeared in Asia and from mid-December 2003 through early August 2004 the outbreaks in poultry caused by the H5N1 virus were reported in nine Asian nations. Russia reports its first H5N1 outbreak in poultry in late July 2005, Kazakhstan and Mongolia in early August. In October 2005, H5N1 was confirmed in poultry in Romania and Turkey. The H5N1 virus was the cause of death or destruction of about 5 billion (milliard) birds. In the current outbreak, laboratory-confirmed human cases were reported in four Asian countries: Cambodia, Indonesia, Thailand and Vietnam The virus infected 126 people and killed 64 of them. According to WHO, FAO and OIE virus H5N1 appearing in many countries and expanding to the West, is a serious threat to human health

Loop-mediated isothermal amplification for the detection of goose circovirus

Abstract Background Goose circovirus (GCV) presents an immunosuppressive problem in production of geese. The infection’s clinical symptoms include growth retardation or feathering disorders but the infection process may remain non-symptomatic what makes the infected birds more susceptible for secondary viral, bacterial and fungal infections. Diagnosis of GCV infection is made by histopathological examination, dot blot hybridization, polymerase chain reaction (PCR) and real-time PCR. However these techniques require application of thermocyclers and qualified staff which may be cost-consuming for some diagnostic units. The aim of this study was to develop loop-mediated isothermal amplification assay (LAMP) as a simple method of GCV detection. Results The presented study has shown LAMP as a rapid tool of detecting DNA of goose circovirus (GCV) as soon in 30 min time. The method used three sets of primers: two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (FL and BL) to accelerate the reaction. The optimum reaction temperature and the time were 61°C for 30 min, respectively. The results were analysed using SYBR Green dye and GelRedTM solutions. Thirty-eight isolates of GCV collected from geese flocks in Poland were examined. For comparison, real-time polymerase chain reaction with F3 and B3 primers and SYBR Green dye was conducted. The obtained results have shown GCV-LAMP as a sensitive, rapid and specific assay and alternative for PCR-based methods. Conclusions The developed technique due to its simplicity may be applied by any veterinary laboratory or even mobile diagnostics units for the routine detection of GCV.</p

Preliminary survey of the occurrence of goose haemorrhagic polyomavirus (GHPV) in wild birds in Poland

Introduction: The aim of the study was to investigate the occurrence of goose haemorrhagic polyomavirus (GHPV) in wild birds inhabiting Poland

Detection of avian reoviruses in wild birds in Poland

Introduction: The purpose of this study was to determine the occurrence of avian reovirus (ARV) infections in wild birds in Poland and attempt to propagate the selected ARV strains in chicken embryo kidney (CEK) cells or chicken SPF embryos. Material and Methods: The study included 192 wild birds representing 32 species, collected between 2014 and 2016. A part of the S4 segment encoding the σNS protein of avian reoviruses (ARVs) isolated from different species of wild birds from that period was amplified. Results: The presence of ARV was demonstrated in 58 (30.2%) wild birds belonging to nine orders. The isolated strains were propagated in chicken embryos by yolk sac inoculation, and CPE was induced in the infected CEK monolayer. Agar gel precipitation showed that two ARV isolates from rock pigeon and mute swan shared a common groupspecific antigen with chicken reovirus S1133. Specific products of predicted size were found in two ARV isolates from the chicken embryo passage and 13 ARVs isolated from CEK cells. Conclusion: The study indicates the high prevalence of ARV among wild birds in Poland and its possible transmission to farmed birds