Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool.We thank Jaime Maldonado and HIPRA (Spain) for the A/Swine/Spain/ 54008/2004 (H3N2) strain, Edward J. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) IAV strain, T. M. Chambers and the University of Kentucky for the A/Equine/OH/1/03 (H3N8) IAV strain, and Hon Ip and the U.S. Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbor Seal/New Hampshire/179629/2011 (H3N8) IAV strains. We thank Sergio López, David Solanes, Francisco X. Abad, Jordi Alberola, Jaume Martorell, and Eduard J. Cunilleras for help in providing different samples and during the experimental infections, as well as the personnel in Cat3 laboratories and the animal house. We thank Adolfo García-Sastre for providing materials and for support as the principal investigator of the NIAID-funded Center for Research in Influenza Pathogenesis (HHSN266200700010C). The research leading to these results received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), the Research Infrastructures Action under grant FP7-228393 (a NADIR project), and projects AGL2010-22200-C02-01 and AGL2007-60274 of the Spanish Ministry of Science and Innovation

Expression dynamics of innate immunity in influenza virus-infected swine

We would like to thank Dr. Jaime Maldonado and HIPRA, Spain for the A/swine/Spain/54008/2004 (H3N2) influenza virus; Dr. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) influenza virus; Dr. Chambers and University of Kentucky for the A/Equine/OH/1/03 (H3N8) influenza virus; and Dr. Hon Ip and the US Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbour Seal/New Hampshire/179629/2011 (H3N8) influenza viruses. The authors thank Sergio López, David Solanes and Francisco X. Abad for their help during the experimental infections as well as the personnel in Cat3 laboratories and animal house. The authors also wish to thank Dr. I. L. Archetti (IZSLER, Brescia, Italy) for the invaluable help in measuring some clinical immunology parameters, Dr. L. Fraile (UdL, Spain) for assistance in statistical analysis, Dr. J. Domínguez (INIA, Spain) for porcine antibodies, Dr. M. Gennari and Dr. M. Giunta (S.S. Genova, IZSPLV, Italy) for assistance in real-time PCR analyses. The skillful technical assistance of Mrs. C. Mantovani (IZSLER, Brescia, Italy) is also gratefully acknowledged. The research leading to these results has received funding from: the European Community's Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228393 (NADIR project), and from the project AGL2010-22200-C02-01 of Spanish Ministry of Science and Innovation.Geological Survey National Wildlife Health Center/[u'duck/Maine/44411-532/2008', u'H3N8']The current circulating swine influenza virus (IV) subtypes in Europe (H1N1, H1N2, and H3N2) are associated with clinical outbreaks of disease. However, we showed that pigs could be susceptible to other IV strains that are able to cross the species barrier. In this work, we extended our investigations into whether different IV strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. For this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a H3N2 Swine IV and four different H3N8 IV strains circulating in different animal species. Pigs had been clinically inspected and four subjects/group were sacrificed at 3, 6, and 21 days post infection. In the present study, all groups but mock exhibited antibody responses to IV nucleoprotein protein. Pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. Interestingly, pigs infected with avian and seal H3N8 strains also showed moderate lesions and viral replication, whereas equine and canine IVs did not cause overt pathological signs, and replication was barely detectable. Swine IV infection induced interferon (IFN)-alpha and interleukin-6 responses in bronchoalveolar fluids (BALF) at day 3 post infection, as opposed to the other non-swine-adapted virus strains. However, IFN-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of IFN-alpha genes. Remarkably, the Equine strain gave rise to a Serum Amyloid A response in BALF despite little if any replication. Each virus strain could be associated with expression of cytokine genes and/or proteins after infection. These responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system

Chlamydia psittaci in feral pigeon: post-mortem, serological and biomolecular aspects.

Scopo del lavoro della presente tesi è stato quello di valutare la prevalenza di Chlamydiaceae, ed in particolare Chlamydia psittaci, in una popolazione aviare con caratteristiche sinantropiche quale il colombo di città (Columba livia var. domestica) dell’areale veneziano. Per evidenziare il patogeno sono state utilizzate metodiche sierologiche, d’isolamento e biomolecolari tradizionali. Contestualmente, mediante tecnologie molecolari innovative, quali microarray ed MLVA (Multilocus VNTR Assay) sono stati valutati i genotipi di C. psittaci e le eventuali altre specie di Chlamydia presenti in tale popolazione. Inoltre, si è proceduto ad una classificazione delle lesioni anatomo-patologiche ed ad una loro correlazione con la presenza del patogeno. I risultati dimostrano che la prevalenza di C. psittaci nella popolazione oggetto dello studio è del 10%. Durante tale studio è stata dimostrata la presenza di un ceppo di C. psittaci atipico, in quanto non classificabile con le attuali tecniche a disposizione. La genotipizzazione dei ceppi di C. psittaci conferma la presenza nel colombo di città del genotipo B, E ed E/B, che solitamente risultano essere coinvolti con minore frequenza in episodi di infezione umana. Inoltre sono stati dimostrati alcuni ceppi classificati come Chlamydia spp., in quanto le metodologie applicate e le conoscenze attuali non permettono ulteriori distinzioni, prospettando la possibilità di un nuovo ceppo. Infine, attraverso l’analisi dei dati raccolti durante la prima fase di campionamenti e successivamente confermati durante la seconda fase, siamo riusciti a strutturare un sistema di selezione, basato su caratteristiche funzionali ed anatomopatologiche, che permette di selezionare in sede necroscopica i colombi molto probabilmente infetti, permettendo conseguentemente una migliore organizzazione e gestione dei campioni di interesse contenendo nel contempo i costi ma mantenendo elevati gli standards diagnostici.The aim of this thesis was to evaluate the prevalence of Chlamydiaceae, in particular of C. psittaci, in sinanthropic birds such as urban pigeons (Columba livia var. domestica) in some areas of Venice. The pathogen was detected with serological, molecular, and traditional culture methods. Innovative molecular tools, such as microarray and MLVA (Multilocus VNTR Assay), were applied in this study in order to evaluate the genotypes of C. psittaci and the other species of Chlamydia present in this avian population to assess the risk of zoonosis posed by pigeons in this urban area. Moreover, we classified and correlated the anatomical and pathological lesions with the pathogen. Our results showed the presence of C. psittaci in urban population of pigeons in Venice, with a prevalence of 10%. We also demonstrated an atypical strain of C. psittaci not yet classified with the available laboratory techniques. Genotyping revealed the presence of genotypes B, E and E/B. This genotypes could be considered less frequently involved in cases of human infection. Additionally, we found other Chlamydia strains suggesting the presence of a new Chlamydia genotype. Finally, the elaboration of the data, collected during the first and second sampling phase, revealed a correlation between C. psittaci and adult females pigeons, presenting hepatomegaly. Based on this results we develop and adopted a diagnostic protocol during necropsy that allows to select pigeons, which have a higher probability to be infected, and a better organization and management of interests samples, containing the economic costs and maintaining high-level of the diagnostic standards

Comparative evaluation of immune responses of swine in PRRS-stable and unstable herds

Porcine Reproductive and Respiratory Syndrome (PRRS) is an elusive model of host/virus relationship in which disease is determined by virus pathogenicity, pig breed susceptibility and phenotype, microbial infectious pressure and environmental conditions. Successful disease control in PRRS-endemic Countries corresponds to "stability", i.e. a condition with no clinical signs of PRRS in the breeding-herd population and no viremia in weaning-age pigs. The aim of this work was to compare the profile and time-course of humoral and cell-mediated immunity in stable and unstable herds, respectively. In particular, we investigated PRRS virus (PRRSV) in serum and group oral fluid samples by Real-time RT-PCR, PRRSV-specific IgA and IgG in oral fluids, serum IgG antibody and the cell-mediated response (PRRSV-specific release of interferon-gamma) in whole blood samples. These parameters were measured in order to identify possible discrepancies in the development and kinetics of the immune response against PRRSV. PRRS-free gilts got regularly infected after entering PRRS-stable and unstable farms. In an open cycle, unstable pig farm PRRSV infection could be demonstrated in all groups of pigs, including suckling piglets. Four main results should be highlighted: A) the precocity of the Ab response in group oral fluids was generally similar to that recorded in sera; B) circulation of PRRSV was consistently detected in all age groups in the unstable herds, as opposed to the stable ones; C) an early, balanced, IgA and IgG response in oral fluids was only observed in the stable herds; D) an early IFN-gamma response after PRRSV infection was often observed in stable herds, as opposed to the unstable ones. These were characterized by IFN-gamma responses in piglets, likely due to transfer of maternal immunity. Most important, the mucosal IgA response was associated with cessation of virus excretion in oral fluid samples of PRRS-unstable herds. The above findings indicate that a peculiar profile of immune response to PRRSV can be found in PRRS-stable herds. Therefore, the outlined immune parameters can represent a useful readout system to evaluate successful adaptation to PRRSV based on acclimatization of breeding animals and management of pig flow

Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool