Species identification of bivalve molluscs by pyrosequencing.

BACKGROUND: The increase in seafood consumption and the presence of different species of bivalves on the global markets has given rise to several commercial frauds based on species substitution. To prevent and detect wilful or unintentional frauds, reliable and rapid techniques are required to identify seafood species in different products. In the present work, a pyrosequencing-based technology has been used for the molecular identification of bivalve species. RESULTS: Processed and unprocessed samples of 15 species belonging to the bivalve families Pectinidae, Mytilidae, Donacidae, Ostreidae, Pharide and Veneridae were analysed and correctly identified by the developed pyrosequencing-based method according to the homology between query sequences of the 16S ribosomal RNA (16S rRNA) and cytochrome c oxidase I (COI) genes and their correspondent reference libraries. This technique exhibits great potential in automated and high-throughput processing systems, allowing the simultaneous analysis of 96 samples in shorter execution and turnaround times. CONCLUSIONS: The correct identification of all the species shows how useful this technique may prove to differentiate species from different products, providing an alternative, simple, rapid and economical tool to detect seafood substitution frauds. © 2016 Society of Chemical Industry.

Influenza A viruses grow in human pancreatic cells and cause pancreatitis and diabetes in an animal model.

Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-α), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals.

Genetic characterization of astroviruses detected in guinea fowl (Numida meleagris) reveals a distinct genotype and suggests cross-species transmission between turkey and guinea fowl.

Astroviruses can infect mammalian and avian species and are often responsible for gastroenteric disease symptoms. In this study, the complete open reading frame (ORF) 2, the 3' end of ORF1b and the corresponding intergenic region of astroviruses identified in farmed guinea fowl (Numida meleagris) were sequenced and genetically analysed. Overall, the genetic sequence of guinea fowl astroviruses was related to turkey astrovirus type 2 (TastV2), although a marked genetic distance was revealed based on ORF2, which might indicate the circulation of a distinct virus genotype and serotype in guinea fowl. Furthermore, the genetic data presented herein suggest that either recombination between different astroviruses infecting distinct hosts or adaptation of a given astrovirus to a new host had occurred. In either case, direct or indirect interspecies transmission of astroviruses is likely to have occurred between turkey and guinea fowl, indicating the ability of viruses belonging to the family Astroviridae to cross species barriers.

Experimental infection of poults and guinea fowl with genetically distinct avian astroviruses.

Avian astroviruses, of the genus Avastrovirus, are recognized as being the cause of enteritis in different bird species worldwide. In particular, turkeys are very susceptible and can be severely affected by this viral agent. More recently, astroviruses were detected in diseased guinea fowl in Italy but whether or not they were the causative agents of the clinical disease was not established. Despite the distribution and relevance of Avastrovirus infection, very little information on pathogenesis or factors influencing the pathogenicity of astroviruses is available. To increase available data on the pathogenesis of these viruses and to test the hypothesis of possible interspecies transmission, experimental infections were carried out in turkeys and guinea fowl with two genetically distinct avian astroviruses, namely TK-6363 and GF-5497, originating respectively from diseased turkey poults and guinea fowl. Data obtained in our study show that both of the viruses selected were able to infect young birds of the species in which they were originally detected. Additionally, these viruses were able to infect young birds of different species causing clinical signs, thus providing experimental evidence for the infection of distinct avian astroviruses in different avian species.

Comparison of the sensitivity, specificity, correlation and inter-assay agreement of eight diagnostic in vitro assays for the detection of African swine fever virus.

With the recent spread of African swine fever (ASF) in Europe, Asia and the Caribbean region, after being endemic for decades in Africa, PCR-based commercial kits and various master mixes are increasingly being used in addition to the Office International des Epizooties-recommended protocol from King et al. (World Organization for Animal Health). Often, the availability and cost of commercial kits or master mixes can be a limiting factor for diagnostic laboratories, in addition to the requirements for transportation and storage of temperature-sensitive reagents in remote areas. In such cases, alternatives should be ready to maximize surveillance and mining of ASF. To evaluate alternatives, we tested five commercial quantitative real-time PCR (qPCR) master mixes from Applied Biosystems, Bio-Rad, Biotechrabbit, Promega and Qiagen using the same primers and probe mix derived from the King et al.'s protocol for the sensitivity, specificity, correlation and inter-assay agreement. We further included three ad hoc molecular diagnostic kits (VetMax™ African Swine Fever Virus Detection Kit [Applied Biosystems], ID Gene African Swine Fever Duplex [ID-Vet] and Virotype ASF PCR Kit [Qiagen/Indical]). The limit of detection (LOD) was assessed for each assay. The comparative study panel comprised 83 archived DNA samples from ASF virus (ASFV) clinical samples, belonging to five different genotypes from outbreaks in 16 countries in Asia and Africa. The analytical specificity was assessed against a panel of swine pathogens. The LOD ranged from 13 to 41 gene copies per reaction; VetMax ™ African Swine Fever Virus Detection Kit from Applied Biosystems exhibited the lowest detection limit (13 gene copies per reaction) and iQ Supermix from Bio-Rad the highest detection limit (41 gene copies per reaction). Cq values obtained from the lowest dilution, in which all replicates (n = 25) could still be amplified (50 gene copies per reaction), were not significantly different between kits using Kruskal-Wallis test. Inter-assay agreement was assessed using statistical test Fleiss-Kappa and was shown to be excellent in all cases. Agreement using statistical test Bland-Altman was good for samples with Cq values <25 and moderate for Cq values >25. We conclude that all the assays evaluated in this study can be used for the routine detection of ASFV.

Lyssavirus detection and typing using pyrosequencing.

Rabies is a fatal zoonosis caused by a nonsegmented negative-strand RNA virus, namely, rabies virus (RABV). Apart from RABV, at least 10 additional species are known as rabies-related lyssaviruses (RRVs), and some of them are responsible for occasional spillovers into humans. More lyssaviruses have also been detected recently in different bat ecosystems, thanks to the application of molecular diagnostic methods. Due to the variety of the members of the genus Lyssavirus, there is the necessity to develop a reliable molecular assay for rabies diagnosis able to detect and differentiate among the existing rabies and rabies-related viruses. In the present study, a pyrosequencing protocol targeting the 3' terminus of the nucleoprotein (N) gene was applied for the rapid characterization of lyssaviruses. Correct identification of species was achieved for each sample tested. Results from the pyrosequencing assay were also confirmed by those obtained using the Sanger sequencing method. A pan-lyssavirus one-step reverse transcription (RT)-PCR was developed within the framework of the pyrosequencing procedure. The sensitivity (Se) of the one-step RT-PCR assay was determined by using in vitro-transcribed RNA and serial dilutions of titrated viruses. The assay demonstrated high analytical and relative specificity (Sp) (98.94%) and sensitivity (99.71%). To date, this is the first case in which pyrosequencing has been applied for lyssavirus identification using a cheaper diagnostic approach than the one for all the other protocols for rapid typing that we are acquainted with. Results from this study indicate that this procedure is suitable for lyssavirus detection in samples of both human and animal origin.

Informing resilience building: FAO's Surveillance Evaluation Tool (SET) Biothreat Detection Module will help assess national capacities to detect agro-terrorism and agro-crime.

Attacks using animal pathogens can have devastating socioeconomic, public health and national security consequences. The livestock sector has some inherent vulnerabilities which put it at risk to the deliberate or accidental spread of disease. The growing concern of countries about the risks of agro-terrorism and agro-crime has led to efforts to prepare against potential attacks. One recent international effort is the launch of a joint OIE, FAO and INTERPOL project in 2019 to build resilience against agro-terrorism and agro-crime targeting animal health with the financial support of the Weapons Threat Reduction Programme of Global Affairs Canada. Given the importance of strong animal health surveillance systems for the early and effective response to agro-terrorism and agro-crime, the project will use the FAO Surveillance Evaluation Tool (SET) and its new Biothreat Detection Module to evaluate beneficiary countries' capacities to detect criminal or terrorist animal health events. This paper presents the development of the new SET Biothreat Detection Module and how it will be used to evaluate surveillance for agro-terrorism and agro-crime animal disease threats. The module will be piloted in early 2021 and, once finalized, will be used by beneficiary countries of the joint OIE-FAO-INTERPOL project. Results from evaluations using SET and its Biothreat Detection Module are expected to provide a baseline from which countries can build targeted capacity for animal disease surveillance including early detection and investigation of potential terrorist or criminal events involving zoonotic and non-zoonotic animal pathogens.

Rabies control in Liberia: Joint efforts towards zero by 30.

Despite declaration as a national priority disease, dog rabies remains endemic in Liberia, with surveillance systems and disease control activities still developing. The objective of these initial efforts was to establish animal rabies diagnostics, foster collaboration between all rabies control stakeholders, and develop a short-term action plan with estimated costs for rabies control and elimination in Liberia. Four rabies diagnostic tests, the direct fluorescent antibody (DFA) test, the direct immunohistochemical test (dRIT), the reverse transcriptase polymerase chain reaction (RT-PCR) assay and the rapid immunochromatographic diagnostic test (RIDT), were implemented at the Central Veterinary Laboratory (CVL) in Monrovia between July 2017 and February 2018. Seven samples (n=7) out of eight suspected animals were confirmed positive for rabies lyssavirus, and molecular analyses revealed that all isolates belonged to the Africa 2 lineage, subgroup H. During a comprehensive in-country One Health rabies stakeholder meeting in 2018, a practical workplan, a short-term action plan and an accurately costed mass dog vaccination strategy were developed. Liberia is currently at stage 1.5/5 of the Stepwise Approach towards Rabies Elimination (SARE) tool, which corresponds with countries that are scaling up local-level interventions (e.g. dog vaccination campaigns) to the national level. Overall an estimated 5.3 - 8 million USD invested over 13 years is needed to eliminate rabies in Liberia by 2030. Liberia still has a long road to become free from dog-rabies. However, the dialogue between all relevant stakeholders took place, and disease surveillance considerably improved through implementing rabies diagnosis at the CVL. The joint efforts of diverse national and international stakeholders laid important foundations to achieve the goal of zero dog-mediated human rabies deaths by 2030.

Pyrosequencing analysis for a rapid classification of fowl adenovirus species.

A rapid fowl adenovirus (FAdV) classification method based on a 30-bp sequence of the hexon loop (L1) was developed using the pyrosequencing technique. FAdV identification is relevant for epidemiological studies and for the adoption of a correct strategy where vaccination is to be used for the control of the disease. FAdV typing is usually performed using polymerase chain reaction coupled with either conventional DNA sequencing or restriction enzyme analysis; however, both methods can be time consuming and/or very expensive to be used as a routine tool. In the present study, polymerase chain reaction and subsequent pyrosequence analysis of the variable hexon L1 region were assessed in order to rapidly differentiate FAdV species. Forty-nine FAdV samples (22 reference strains and 27 field isolates) were tested and the results were compared with those obtained by conventional DNA sequencing. The results clearly demonstrated that pyrosequence analysis provides a new approach for a rapid differentiation and classification of the FAdV species that is faster, more cost-effective and easier to interpret than other techniques commonly used.

An inter-laboratory trial as a tool to increase rabies diagnostic capabilities of Sub-Saharan African Veterinary laboratories.

To achieve the goal of eliminating dog-mediated human rabies deaths by 2030, many African countries have agreed to list rabies as a priority zoonotic disease and to undertake both short and long-term control programs. Within this context, reliable local diagnosis is essential for the success of field surveillance systems. However, a harmonized, sustainable and supportive diagnostic offer has yet to be achieved in the continent. We herewith describe the organization and outcome of a proficiency test (PT) for the post-mortem diagnosis of rabies in animals, involving thirteen veterinary laboratories and one public health laboratory in Africa. Participants were invited to assess both the performance of the Direct Fluorescent Antibody (DFA) test and of a conventional RT-PCR. From the submitted results, while thirteen laboratories proved to be able to test the samples through DFA test, eleven performed the RT-PCR method; ten applied both techniques. Of note, the number of laboratories able to apply rabies RT-PCR had increased from four to ten after the exercise. Importantly, results showed a higher proficiency in applying the molecular test compared to the DFA test (concordance, sensitivity and specificity: 98.2%, 96.97% and 100% for RT-PCR; 87.69%, 89.23% and 86.15% for DFA test), indicating the feasibility of molecular methods to diagnose animal pathogens in Africa. Another positive outcome of this approach was that negative and positive controls were made available for further in-house validation of new techniques; in addition, a detailed questionnaire was provided to collect useful and relevant information on the diagnostic procedures and biosafety measures applied at laboratory level.

Development and validation of a one-step real-time PCR assay for simultaneous detection of subtype H5, H7, and H9 avian influenza viruses.

Among the different hemagglutinin (HA) subtypes of avian influenza (AI) viruses, H5, H7, and H9 are of major interest because of the serious consequences for the poultry industry and the increasing frequency of direct transmission of these viruses to humans. The availability of new tools to rapidly detect and subtype the influenza viruses can enable the immediate application of measures to prevent the widespread transmission of the infection. In this study, a novel one-step real-time reverse transcription-PCR (RRT-PCR) was developed to detect simultaneously the H5, H7, and H9 subtypes of AI viruses from clinical samples of avian origin. The sensitivity of the RRT-PCR assay was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection ranging from 10(1) to 10(3) RNA copies and from 10(1) 50% egg infectious dose (EID(50))/100 microl to 10(2.74) EID(50)/100 microl with titrated viruses. Excellent results were achieved in the intra- and interassay variability tests. The comparison of the results with those obtained from the analysis of 725 avian samples by means of the reference method (virus isolation [VI]) showed a high level of agreement. To date, this is the first real-time PCR protocol available for the simultaneous detection of AI viruses belonging to subtypes H5, H7, and H9, and the results obtained indicate that this method is suitable as a routine laboratory test for the rapid detection and differentiation of the three most-important AI virus subtypes in samples of avian origin.

Study of 2009 H1N1 Pandemic Influenza Virus as a Possible Causative Agent of Diabetes.

Context: Recent studies have suggested that influenza A virus (IAV) might be involved in the etiology of diabetes. Objective and Methods: To address this question, we tested the ability of H1N1 pandemic IAV to infect, replicate, and damage human ß cells/pancreatic islets in vitro and induce pancreatic damage and/or glucose metabolism alterations in chemical and autoimmune models of ß cell damage in vivo. Moreover, we looked for direct and/or indirect evidence of correlation between IAV infection and autoimmunity/diabetes in humans. Results: Human H1N1 A/California/2009-derived viruses infected human pancreatic islets in vitro, inducing a proinflammatory response associated with substantial increases of CXCL9 and CXCL10 release. In vivo, infected mice showed a clear susceptibility to the virus, with its localization also found in extrapulmonary organs, including the pancreas. Infection was able to induce mild modifications of glycemia in C57B6 mice after chemical damage of islets but did not modulate the autoimmune damage of islets in NOD mice. One of 69 nasopharyngeal swabs collected from patients at the onset of type 1 diabetes yielded positive results for IAV. Pancreas sections from 17 organ donors available from the Network for Pancreatic Organ Donors With Diabetes showed the persistence of CXCL10-positive cells in islet autoimmunity-positive subjects; however, extremely rare cells stained for viral RNA and not preferentially in autoimmune subjects. Conclusion: Influenza H1N1 pdm strains are able to infect and replicate in mammalian pancreatic cells both in vitro and in vivo but did not cause any functional impairment consistent with diabetes.

Pyrosequencing as a tool for rapid fish species identification and commercial fraud detection.

The increased consumption of fish products, as well as the occurrence of exotic fish species in the Mediterranean Sea and in the fish market, has increased the risk of commercial fraud. Furthermore, the great amount of processed seafood products has greatly limited the application of classic identification systems. DNA-based identification allows a clear and unambiguous detection of polymorphisms between species, permitting differentiation and identification of both commercial fraud and introduction of species with potential toxic effects on humans. In this study, a novel DNA-based approach for differentiation of fish species based on pyrosequencing technology has been developed. Raw and processed fish products were tested, and up to 25 species of fish belonging to Clupeiformes and Pleuronectiformes groups were uniquely and rapidly identified. The proper identification based on short and unique genetic sequence signatures demonstrates that this approach is promising and cost-effective for large-scale surveys.

Rapid pathotyping of Newcastle Disease Virus by pyrosequencing.

Newcastle Disease Virus (NDV) is the only member of serotype 1 avian paramyxoviruses (APMV-1) that causes respiratory and neurological disease in chickens and other species of birds and can cause severe economic losses in the poultry sector. Due to the relevant variability of the genome and the pathogenicity of NDV isolates, their detection in a specimen is not sufficient to provide and confirm an exact diagnosis, and so the assessment of virus pathotype is required. To diagnose rapidly and pathotype NDV directly in clinical specimens, a method based on RT-PCR and pyrosequencing analysis has been developed and is reported in the present study. A pair of degenerated primers was designed to amplify a portion of the fusion (F) gene responsible for virulence and used to test 315 specimens collected from 2006 to 2011. The subsequent pyrosequencing reaction identified a 30-bp region encompassing the cleavage site. A total of 213 out of 315 samples were pyrosequenced and results were compared and confirmed by the Sanger sequencing procedure, which is traditionally performed for NDV pathotyping. The pyrosequencing reaction provided high quality results in real time and proved to be more rapid and cost-efficient than the classical sequencing procedure, indicating it as a possible valid alternative to the currently used diagnostic assays for NDV.

Pyrosequencing of the rabies virus glycoprotein gene to demonstrate absence of vaccine-associated rabies cases following oral vaccination.

Replication competent vaccines have been used successfully for the control of terrestrial rabies, mainly in wildlife; however, these vaccine strains occasionally may induce rabies. In this study, a pyrosequencing protocol for the rapid identification of vaccine-associated rabies viruses was applied to the 2008-2011 Italian epidemic. There was no evidence of vaccine-associated rabies cases following oral vaccination of foxes with the SAG2 and SADB19 vaccine strains.

Prevalence and risk factors of astrovirus infection in puppies from French breeding kennels.

Aiming at determining the prevalence and the risk factors associated to astrovirus infection in puppy, fecal samples were collected in 316 puppies (age from 5 to 14 weeks of age) from 33 French breeding kennels. Data were registered for each puppy, including age, breed, gender, origin of the dog, and feces quality. The samples were tested by specific RT-PCR for the presence of canine astrovirus. Astroviruses were identified in 20.9% (66/316) of the puppies and in 42% (14/33) of the breeding kennels. Young puppies (i.e. <7 weeks of age) and puppies from large breeding kennels were more likely to be infected by the astrovirus. No association between the quality of feces and astrovirus infection could be determined in this survey.

Genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea.

Astroviruses have been described in several animals species frequently associated with diarrhoea, especially in young animals. In dogs, astrovirus-like particles have been observed sporadically and very little is known about their epidemiology and characteristics. In this paper, we describe the detection of astrovirus-like particles in symptomatic puppies. Furthermore, for the first time in this species, the presumptive identification made by electron microscopy was confirmed by genetic analysis of the viral RNA conducted directly on the clinical specimens. Genetic sequences of ORF2 (2443 nt), encoding for the capsid protein, and partial sequence of ORF1b (346 nt), encoding for the viral polymerase, identified the viruses as member of the family Astroviridae. The phylogenetic analysis clearly clustered canine astroviruses in the genus Mamastrovirus. Relative closest similarities were revealed with a cluster comprising human, porcine and feline astroviruses, based on the ORF2 sequences available. Based on the species definition for astroviruses and on the data obtained in this study, we suggest a new species of astrovirus - canine astrovirus, CaAstV - to be included in the genus Mamastrovirus.