Rapid, Sensitive, and Species-Specific Detection of Conventional and Recombinant Herpesvirus of Turkeys Vaccines Using Loop-Mediated Isothermal Amplification Coupled With a Lateral Flow Device Readout

Marek's disease, an economically important disease of chickens caused by virulent serotype 1 strains of the Mardivirus Marek's disease virus (MDV-1), is effectively controlled in the field by live attenuated vaccine viruses including herpesvirus of turkeys (HVT)-both conventional HVT (strain FC126) and, in recent years, recombinant HVT viruses carrying foreign genes from other avian viruses to protect against both Marek's disease and other avian viral diseases. Testing to monitor and confirm successful vaccination is important, but any such test must differentiate HVT from MDV-1 and MDV-2, as vaccination does not prevent infection with these serotypes. End-point and real-time PCR tests are widely used to detect and differentiate HVT, MDV-1 and MDV-2 but require expensive specialist laboratory equipment and trained operators. Here, we developed and validated two tube-based loop-mediated isothermal amplification tests coupled with detection by lateral flow device readout (LAMP-LFD): an HVT-specific test to detect both conventional and recombinant HVT strains, and a second test using novel LAMP primers to specifically detect the Vaxxitek (R) recombinant HVT. Specificity was confirmed using DNA extracted from virus-infected cultured cells, and limit of detection was determined using plasmid DNA carrying either the HVT or Vaxxitek (R) genome. The LAMP-LFD tests accurately detected all HVT vaccines, or Vaxxitek (R) only, in crude DNA as well as purified DNA extracted from field samples of organs, feathers, or poultry house dust that were confirmed positive for HVT by real-time PCR. These LAMP-LFD tests have potential for specific, rapid, simple, and inexpensive detection of HVT vaccines in the field

Molecular detection and characterization of Mycoplasma gallisepticum and Mycoplasma synoviae strains in backyard poultry in Italy

ABSTRACT Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) represent the most important avian Mycoplasma species in the poultry industry, causing considerable economic losses. In Italy, the presence of MG or MS has been investigated especially in commercial poultry farms. To our knowledge, no systematic investigations on MG or MS presence using highly specific diagnostic assays have been performed in backyard poultry. The aim of this study was to detect and molecularly characterize MG and MS strains in 11 backyard poultry flocks located in different regions of Italy. Tracheal swabs were collected and DNA was extracted. For MS, a PCR targeting a vlhA gene fragment was performed, and typing and subtyping was attempted. The presence of MG was investigated by a screening PCR, then MG typing by gene-targeted sequencing (GTS). All the amplicons were sequenced, then MG and MS dendrograms were constructed. All the flocks examined resulted Mycoplasma positive: 5 out of 11 (45.45%) were MG and MS positive, 3 (27.27%) were MG positive, and the remaining 3 (27.27%) were MS positive. The MS detections were assigned to types C, D, and F. All strains of type D belonged to subtype D1 and 2 unknown subtypes were identified. A MS sequence showed peculiar characteristics, which did not allow assignment to a known MS type or subtype. MG GTS analysis identified 6 MG strains belonging to 5 subclusters circulating in Italian backyards chicken flocks. The results of this study provide evidence of a risk for commercial poultry farms, especially in areas where backyard and commercial farms are close, suggesting the implementation of biosecurity measures

Molecular characterization of the meq gene of Marek's disease viruses detected in unvaccinated backyard chickens reveals the circulation of low- and high-virulence strains

ABSTRACT Marek's disease (MD) is an important lymphoproliferative disease of chickens, caused by Gallid alphaherpesvirus 2 (GaHV-2). Outbreaks are commonly reported in commercial flocks, but also in backyard chickens. Whereas the molecular characteristics of GaHV-2 strains from the commercial poultry sector have been reported, no recent data are available for the rural sector. To fill this gap, 19 GaHV-2 strains detected in 19 Italian backyard chicken flocks during suspected MD outbreaks were molecularly characterized through an analysis of the meq gene, the major GaHV-2 oncogene. The number of four consecutive prolines (PPPP) within the proline-rich repeats of the Meq transactivation domain, the proline content, and the presence of amino acid (aa) substitutions were determined. Phylogenetic analysis was performed using the Maximum Likelihood method. Sequence analysis revealed a heterogeneous population of GaHV-2 strains circulating in Italian backyard flocks. Seven strains, detected from birds affected by classical MD, showed a unique meq isoform of 418 aa with a very high number of PPPP motifs. Molecular and clinical features are suggestive of a low oncogenic potential of these strains. The remaining 12 strains, detected from flocks experiencing acute MD, transient paralysis, or sudden death, had shorter Meq protein isoforms (298 or 339 aa) with a lower number of PPPP motifs and point mutations interrupting PPPP. These features allow us to assert the high virulence of these strains. These findings reveal the circulation of low- and high-virulence GaHV-2 strains in the Italian rural sector

avian metapneumovirus circulation in italian broiler farms

Abstract With increasing frequency, avian Metapneumovirus (aMPV) is reported to induce respiratory signs in chickens. An adequate knowledge of current aMPV prevalence among Italian broilers is lacking, with little information available on its economical and health impact on the poultry industry. In order to collect preliminary data on the epidemiological context of aMPV in broiler flocks, a survey was performed in areas of Northern Italy with high poultry density from 2014 to 2016. Upper respiratory tract swabs were collected and processed by A and B subtype-specific multiplex real-time reverse transcription PCR (RT-PCR). Samples were also screened for infectious bronchitis virus (IBV) by generic RT-PCR and sequencing. Productive data and respiratory signs were detailed where possible. The high prevalence of aMPV was confirmed in broilers older than 26 d and also attested in IBV-negative farms. All aMPV detections belonged to subtype B. Italian strain genetic variability was evaluated by the partial attachment (G) gene sequencing of selected strains and compared with contemporary turkey strains and previously published aMPV references, revealing no host specificity and the progressive evolution of this virus in Italy

Insights on epidemiology and control of Marek’s disease through traditional and novel molecular tools

Marek's disease (MD) is a contagious, lymphoproliferative and neuropathic disease of poultry caused by a ubiquitous lymphotropic and oncogenic virus, Gallid alphaherpesvirus 2 (GaHV-2). MD has been reported in all poultry-rearing countries and is among the viral diseases with the highest economic impact in the poultry industry worldwide, including Italy. MD has been also recognized as one of the leading causes of mortality in backyard poultry. The present doctoral thesis aimed at exploring Marek's disease virus molecular epidemiology in Italian commercial and backyard chicken flocks and, for the first time, in commercial turkeys affected by clinical MD. Molecular biology techniques targeting the full-length meq gene, the major GaHV-2 oncogene, were used to detect and characterize the circulating GaHV-2 strains searching for genetic markers of virulence. A final study focused on the development of rapid, sensitive, and species-specific loop-mediated isothermal amplification assays coupled with a lateral flow device readout for the detection of conventional and recombinant HVT-based vaccines is included in the thesis. HVT vaccines, currently used to protect chickens from MD, are referred to as "leaky", as they do not impede the infection, replication, and shedding of field GaHV-2: vaccinal and field viruses can coexist in the vaccinated host and molecular tests able to discriminate between GaHV-2 and HVT are required. These new simple, fast, and accurate tests for the monitoring of MD vaccination success in the field could be greatly beneficial for field veterinarians, small laboratories, and more broadly for resource-limited settings

Genetic Heterogeneity among Chicken Infectious Anemia Viruses Detected in Italian Fowl

Chicken infectious anemia virus (CIAV) is a pathogen of chickens associated with immunosuppression and with a disease named chicken infectious anemia. The present survey reports an epidemiological study on CIAV distribution in Italian broiler, broiler breeder and backyard chicken flocks. Twenty-five strains were detected by a specifically developed nested PCR protocol, and molecularly characterized by partial VP1 gene or complete genome sequencing. Viral DNA amplification was successfully obtained from non-invasive samples such as feathers and environmental dust. Sequence and phylogenetic analysis showed the circulation of field or potentially vaccine-derived strains with heterogeneous sequences clustered into genogroups II, IIIa, and IIIb. Marker genome positions, reported to be correlated with CIAV virulence, were evaluated in field strains. In conclusion, this is the first survey focused on the molecular characteristics of Italian CIAVs, which have proved to be highly heterogeneous, implementing at the same time a distribution map of field viruses worldwide

Analysis of provincial and municipal regulations governing crustacean welfare in Italy

The trade in live crustaceans implies keeping these animals alive after capture and/or farming until purchase by the final consumer. Regarding animal welfare, the European Union includes cephalopods in Directive 2010/63/EU on the protection of animals used for scientific purposes, but there are no further regulations on crustaceans in EU legislation. The present study analysed the provisions of Italian municipal regulations on animal welfare applicable to crustaceans. Only 62 of the 110 municipal websites of the provincial capitals reported a regulation safeguarding animal welfare. These regulations contain different rules on: aquaria characteristics (size, volume and shape); management of aquaria; maintenance (preservation and exposure) of live aquatic species; slaughtering and/or suppression of aquatic species and crustaceans; tying of crustacean claws; and crustacean cooking. The analysis on Italian municipal regulations on crustaceans\u2019 animal welfare showed that the provisions are vague, lacking uniformity and scientific guidelines

Mislabelling of Döner kebab sold in Italy

The aim of this study was to assess whether Döner kebabs sold in Italy comply with legal labelling requirements. A checklist of EU labelling requirements was drawn up and nine labels of Döner kebab marketed in the province of Modena were collected to evaluate the mandatory information laid down in article 9 of Regulation (EU) No 1169/2011 together with the mandatory requirements of Regulation (EC) 853/2004 for meat preparations. The study showed that different additives are commonly used in Döner kebabs, including E 621 (monosodium glutamate). This additive is not included among the ingredients authorized for the meat preparation food category. Eight frozen Döner kebab samples were taken and a visual assessment and a histologic examination were performed to verify potential changes in the meat’s inner muscle fibre structure that could allow the product to be considered among meat preparations or meat products. The Döner kebab labels showed several systematically deficient regulatory requirements in both extrinsic defects (not related to food composition) and aspects concerning the protection of consumers’ health. Histological examination showed that the tissue was moderately swollen, but the muscle fibre structure was intact. Kebab can be reasonably classified as a meat preparation thereby precluding the use monosodium glutamate (E 621) as an ingredient

Molecular characterization of Marek\u2019s disease virus detected in backyard chickens affected by classical nervous form

An outbreak of Marek\u2019s disease nervous form in unvaccinated Cochin chickens from an Italian backyard flock is reported. After suppression two birds, showing inability to stand upright and spastic paralysis of legs, were necropsied and organs were taken for histologic examination and molecular analysis. MDV-1 was detected from feathers by PCR targeting meq gene.meq gene of the detected strain was fully sequenced along with commercial vaccines most commonly used in Italy. Nucleotide and amino acid sequences were compared to selected MDV-1 reference strains with different degrees of pathogenicity, retrieved from GenBank. The sequence analysis and the high number of PPPP repeats found in the transactivation domain of the Meq protein showed that the detected virus was closely related to mild or attenuated MDV-1 strains, but different from the analyzed vaccines. The molecular results were supported by clinical, macroscopic and microscopic findings. A- and B-type lesions, highly suggestive of a mild MDV-1 infection, were observed in peripheral nerves.MHC haplotype B19, known to be correlated with high susceptibility to Marek\u2019s disease, was found in affected chickens by PCR and sequencing of B-L\u3b2II family genes. Because sequencing of the meq gene is not a sufficient criteria in determining the pathotype, virus isolation will be attempted in order to confirm the molecular results by in vivo experimental trials