Improved diagnostics and surveillance identify novel reassortant swine influenza A viruses in Chile

Influenza A Virus (IAV) circulates endemically in nature, representing a constant concern to public health and animal production systems worldwide. The emergence of the 2009 H1N1 influenza A pandemic (A(H1N1)pdm09) highlighted the need of conducting systematic surveillance studies, and evidenced the significant gap of knowledge of the swine IAVs (SwIAV) circulating in pigs worldwide at the time of the outbreak. This also highlighted the role of swine as important reservoirs for the generation of endemic strains with zoonotic and pandemic potential. Despite this, comprehensive information of SwIAV circulating in Latin America is still lacking. While Chile has strong ecological barriers and high stringency controls regarding livestock trading, IAV is endemic and has been consistently detected in pigs. This has caused great concerns for the swine industry, because of the on-going clinical and production losses. Nevertheless, few surveillance efforts have been conducted in Chile and there is limited information regarding the genetic diversity and origin of swIAVs. Determining the viral subtypes and genotypes circulating in swine is key for the development of appropriate vaccine approaches and control measures for the swine industry, and it is crucial for identifying novel strains with pandemic potential. We developed improved diagnostic tools to detect endemic viruses and characterized the diversity and origin of SwAIV and its prevalence in Chilean swine production farms. From December 2013 to June 2015, we sampled 27 farms representative of intensive swine production systems located between the Valparaiso and Araucania administrative regions. Animals were mainly found to be susceptible to IAV infection at 50 to 110 days of age. Serological analysis of 718 serum samples obtained from 3 different farms showed 91-59% prevalence to SwIAV, with 18-67% positivity to A(H1N1)pdm09-like strains and 29-58% positivity to a novel SwH1N2 virus. We also obtained a total of 1016 nasal swabs (NS, 85%) and 176 oral fluids (OF, 15%) that were tested by Real-time RT-PCR (qPCR) and virus isolation. Of these, 295 samples (25%) were positive to swIAV (23% of NS and 38% of OF). A large number of farms (21 out of 27, 78%) were positive in at least one visit, and most farms showed temporal co-circulation of at least 2 viruses or mixed infections. We subtyped selected positive samples and sequenced 51 complete viral genomes using the Illumina platform. Nineteen additional hemagglutinins (HA) genes were sequenced by Sanger. We performed Bayesian Evolutionary Analyses to reconstruct the phylogenies of the viral segments. This identified the circulation of 2 predominant swIAV genotypes in Chile, the A(H1N1)pdm09-like (35%) strain and a novel SwH1N2 virus (45%). This H1N2 virus is unique to Chile since it is genetically distinct from the H1 virus clusters seen in North America and is not related to any previously reported IAV. The genome of this swH1N1 virus contains genes from 3 different human contemporary viruses. Its H1 and N2 genes are derived from human H1N1 and H3N2 viruses from the mid 90’s, respectively, suggesting that both of these human viruses were likely introduced into the Chilean swine population during that time. All the internal genes are from the A(H1N1)pdm09 strain, indicating that multiple and recent reassortment events gave rise to this novel reassortant virus. Of interest, we identified additional reassortant viruses that also contain the internal genes derived from the A(H1N1)pdm09 strain. These included an H1N1 containing the novel SwH1 and NA gene derived from the A(H1N1)pdm09-like strain, an H1N2 virus containing an H1 derived from the A(H1N1)pdm09 strain and the novel SwN2, a SwH3N2 virus and a human-like H3N2 virus, among others. Additionally, our analyses also revealed at least 3 independent human-to-swine introductions of the A(H1N1)pdm09 strain within the last 4 years in Chile. Overall this indicates that close human-swine interactions greatly contribute to the genetic diversity and emergence of IAVs in Chilean swine. This is the first comprehensive molecular epidemiological study of swIAV in Chile demonstrating the co-circulation of multiple viral strains in intensive swine production systems. Our data emphasizes the value of conducting long-term SwIAV surveillance in Latin America, a poorly studied region of the world. Acknowledgements: CEIRS program: HHSN266200700010C, HHSN272201400008C from NIH-NIAID, FONDEF IDeA Grant ID14I10201 and Proyecto Anillo de Investigación en Ciencia y Tecnología - PIA ACT1408, both from CONICYT Chile

Evaluation of potential risk of transmission of avian influenza A viruses at live bird markets in response to unusual crow die-offs in Bangladesh

In response to unusual crow die‐offs from avian influenza A(H5N1) virus infection during January‐February 2017 in Dhaka, Bangladesh, a One Health team assessed potential infection risks in live bird markets (LBMs). Evidence of aerosolized avian influenza A viruses was detected in LBMs and in the respiratory tracts of market workers, indicating exposure and potential for infection. This study highlighted the importance of surveillance platforms with a coordinated One Health strategy to investigate and mitigate zoonotic risk

Efecto de la invasión de acacia negra (<i>Gleditsia triacanthos</i> L.) sobre arroyos pampeanos

Pampean fluvial ecosystems of low order usually lack of riparian vegetation. In recent years the invasion of honey locust (Gleditsia triacanthos L.) has caused changes in landscape composition. The aim of this study was to evaluate the effect of honey locust invasion on pampean streams. Three reaches with different degree of invasion were selected and compared with three control reaches with no canopy. In order to evaluate the effect of invasion, nutrients (soluble reactive phosphorus, ammonium and nitrates), biomass of producers (seston, macrophytes, periphyton and phytobenthos) and net production in each reach were determined. Significant differences were observed for all of variables estimated between reaches with and without canopy, but not in all cases. The amount of variables with significant differences between both reaches rises following a gradient of increasing invasion levels. Furthermore, differences are more pronounced with increasing percentage of honey locust coverage on the stream. We conclude that sustained invasion of honey locust modifies the concentration of nutrients in water, the biomass of some communities and the production of these communities. This effect could be avoided with early management of the invasion of honey locust.Facultad de Ciencias Naturales y Muse

Efecto de la invasión de acacia negra (<i>Gleditsia triacanthos</i> L.) sobre arroyos pampeanos

Pampean fluvial ecosystems of low order usually lack of riparian vegetation. In recent years the invasion of honey locust (Gleditsia triacanthos L.) has caused changes in landscape composition. The aim of this study was to evaluate the effect of honey locust invasion on pampean streams. Three reaches with different degree of invasion were selected and compared with three control reaches with no canopy. In order to evaluate the effect of invasion, nutrients (soluble reactive phosphorus, ammonium and nitrates), biomass of producers (seston, macrophytes, periphyton and phytobenthos) and net production in each reach were determined. Significant differences were observed for all of variables estimated between reaches with and without canopy, but not in all cases. The amount of variables with significant differences between both reaches rises following a gradient of increasing invasion levels. Furthermore, differences are more pronounced with increasing percentage of honey locust coverage on the stream. We conclude that sustained invasion of honey locust modifies the concentration of nutrients in water, the biomass of some communities and the production of these communities. This effect could be avoided with early management of the invasion of honey locust.Facultad de Ciencias Naturales y Muse

In vivo testing of novel vaccine prototypes against Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a Gram-negative bacterium that represents the main cause of porcine pleuropneumonia in pigs, causing significant economic losses to the livestock industry worldwide. A. pleuropneumoniae, as the majority of Gram-negative bacteria, excrete vesicles from its outer membrane (OM), accordingly defined as outer membrane vesicles (OMVs). Thanks to their antigenic similarity to the OM, OMVs have emerged as a promising tool in vaccinology. In this study we describe the in vivo testing of several vaccine prototypes for the prevention of infection by all known A. pleuropneumoniae serotypes. Previously identified vaccine candidates, the recombinant proteins ApfA and VacJ, administered individually or in various combinations with the OMVs, were employed as vaccination strategies. Our data show that the addition of the OMVs in the vaccine formulations significantly increased the specific IgG titer against both ApfA and VacJ in the immunized animals, confirming the previously postulated potential of the OMVs as adjuvant. Unfortunately, the antibody response raised did not translate into an effective protection against A. pleuropneumoniae infection, as none of the immunized groups following challenge showed a significantly lower degree of lesions than the controls. Interestingly, quite the opposite was true, as the animals with the highest IgG titers were also the ones bearing the most extensive lesions in their lungs. These results shed new light on A. pleuropneumoniae pathogenicity, suggesting that antibody-mediated cytotoxicity from the host immune response may play a central role in the development of the lesions typically associated with A. pleuropneumoniae infections

Effects of the herbicide Roundup on freshwater microbial communities: a mesocosm study

The impact of the widely used herbicide glyphosate has been mainly studied in terrestrial weed control, laboratory bioassays, and field studies focusing on invertebrates, amphibians, and fishes. Despite the importance of phytoplankton and periphyton communities at the base of the aquatic food webs, fewer studies have investigated the effects of glyphosate on freshwater microbial assemblages. We assessed the effect of the commercial formulation Roundup using artificial earthen mesocosms. The herbicide was added at three doses: a control (without Roundup) and two treatments of 6 and 12 mg/L of the active ingredient (glyphosate). Estimates of the dissipation rate (k) were similar in the two treatments (half-lives of 5.77 and 7.37 d, respectively). The only two physicochemical parameters showing statistically significant differences between treatments and controls were the downward vertical spectral attenuation coefficient kd(k), where k is wavelength, and total phosphorus concentration (TP). At the end of the experiment, the treated mesocosms showed a significant increase in the ratio kd(490 nm)/kd(550 nm) and an eightfold increase in TP. Roundup affected the structure of phytoplankton and periphyton assemblages. Total micro- and nanophytoplankton decreased in abundance in treated mesocosms. In contrast, the abundance of picocyanobacteria increased by a factor of about 40. Primary production also increased in treated mesocosms (roughly by a factor of two). Similar patterns were observed in the periphytic assemblages, which showed an increased proportion of dead : live individuals and increased abundances of cyanobacteria (about 4.5-fold). Interestingly, the observed changes in the microbial assemblages were captured by the analysis of the pigment composition of the phytoplankton, the phytoplankton absorption spectra, and the analysis of the optical properties of the water. The observed changes in the structure of the microbial assemblages are more consistent with a direct toxicological effect of glyphosate rather than an indirect effect mediated by phosphorus enrichment.Facultad de Ciencias Naturales y Muse

Efecto de la invasión de acacia negra (<i>Gleditsia triacanthos</i> L.) sobre arroyos pampeanos

Pampean fluvial ecosystems of low order usually lack of riparian vegetation. In recent years the invasion of honey locust (Gleditsia triacanthos L.) has caused changes in landscape composition. The aim of this study was to evaluate the effect of honey locust invasion on pampean streams. Three reaches with different degree of invasion were selected and compared with three control reaches with no canopy. In order to evaluate the effect of invasion, nutrients (soluble reactive phosphorus, ammonium and nitrates), biomass of producers (seston, macrophytes, periphyton and phytobenthos) and net production in each reach were determined. Significant differences were observed for all of variables estimated between reaches with and without canopy, but not in all cases. The amount of variables with significant differences between both reaches rises following a gradient of increasing invasion levels. Furthermore, differences are more pronounced with increasing percentage of honey locust coverage on the stream. We conclude that sustained invasion of honey locust modifies the concentration of nutrients in water, the biomass of some communities and the production of these communities. This effect could be avoided with early management of the invasion of honey locust.Facultad de Ciencias Naturales y Muse

Histopathological and immunohistochemical findings of swine with spontaneous influenza A infection in Brazil, 2009-2010

Swine influenza (SI) is caused by the type A swine influenza virus (SIV). It is a highly contagious disease with a rapid course and recovery. The major clinical signs and symptoms are cough, fever, anorexia and poor performance. The disease has been associated with other co-infections in many countries, but not in Brazil, where, however, the first outbreak has been reported in 2011. The main aim of this study was to characterize the histological features in association with the immunohistochemical (IHC) results for influenza A (IA), porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) in lung samples from 60 pigs submitted to Setor de Patologia Veterinária at the Universidade Federal do Rio Grande do Sul (SPV-UFRGS), Brazil, during 2009-2010. All of these lung samples had changes characterized by interstitial pneumonia with necrotizing bronchiolitis, never observed previously in the evaluation of swine lungs in our laboratory routine. Pigs in this study had showed clinical signs of a respiratory infection. Swine samples originated from Rio Grande do Sul 31 (52%), Santa Catarina 14 (23%), Paraná 11 (18%), and Mato Grosso do Sul 4 (7%). Positive anti-IA IHC labelling was observed in 45% of the cases, which were associated with necrotizing bronchiolitis, atelectasis, purulent bronchopneumonia and hyperemia. Moreover, type II pneumocyte hyperplasia, alveolar and bronchiolar polyp-like structures, bronchus-associated lymphoid tissue (BALT) hyperplasia and pleuritis were the significant features in negative anti-IA IHC, which were also associated with chronic lesions. There were only two cases with positive anti-PCV2 IHC and none to PRRSV. Therefore, SIV was the predominant infectious agent in the lung samples studied. The viral antigen is often absent due to the rapid progress of SI, which may explain the negative IHC results for IA (55%); therefore, IHC should be performed at the beginning of the disease. This study has shown how important a careful histological evaluation is for the diagnosis. Since 2009, a new histological feature of swine pneumonia in animals with respiratory clinical signs has been observed in samples from pigs with clinical respiratory disease submitted to SPV-UFRGS. In addition, the results proved the importance of histological evaluation for swine herd health management