Update on Glässer’s disease: How to control the disease under restrictive use of antimicrobials

Antimicrobials have been commonly used to control bacterial diseases in farm animals. The efficacy of these drugs deterred the development of other control measures, such as vaccines, which are currently getting more attention due to the increased concern about antimicrobial resistance. Glässer’s disease is caused by Glaesserella (Haemophilus) parasuis and affects pork production around the world. Balance between colonization and immunity seems to be essential in disease control. Reduction in antimicrobial use in veterinary medicine requires the implementation of preventive measures, based on alternative tools such as vaccination and other strategies to guarantee a beneficial microbial colonization of the animals. The present review summarizes and discusses the current knowledge on diagnosis and control of Glässer’s disease, including prospects on alternatives to antimicrobials.info:eu-repo/semantics/publishedVersio

Haemophilus parasuis VtaA2 is involved in adhesion to extracellular proteins

Haemophilus parasuis is part of the microbiota of the upper respiratory tract in swine. However, virulent strains can cause a systemic disease known as Glässer’s disease. Several virulence factors have been described in H. parasuis including the virulence-associated trimeric autotransporters (VtaAs). VtaA2 is up-regulated during infection and is only found in virulent strains. In order to determine its biological function, the vtaA2 gene was cloned with its native promotor region in pACYC184, and the transformed Escherichia coli was used to perform functional in vitro assays. VtaA2 was found to have a role in attachment to plastic, mucin, BSA, fibronectin and collagen. As other VtaAs from H. parasuis, the passenger domain of VtaA2 contains collagen domains. In order to examine the contribution of the collagen repeats to VtaA2 function, a recombinant vtaA2 without the central collagen domains was obtained and named vtaA2OL. VtaA2OL showed similar capacity than VtaA2 to adhere to plastic, mucin, BSA, fibronectin and plasma but a reduced capacity to adhere to collagen, suggesting that the collagen domains of VtaA2 are involved in collagen attachment. No function in cell adhesion and invasion to epithelial alveolar cell line A549 or unspecific binding to primary alveolar macrophages was found. Likewise VtaA2 had no role in serum or phagocytosis resistance. We propose that VtaA2 mediates adherence to the host by binding to the mucin, found in the upper respiratory tract mucus, and to the extracellular matrix proteins, present in the connective tissue of systemic sites, such as the serosa.info:eu-repo/semantics/publishedVersio

Haemophilus parasuis VtaA2 is involved in adhesion to extracellular proteins

International audienceAbstractHaemophilus parasuis is part of the microbiota of the upper respiratory tract in swine. However, virulent strains can cause a systemic disease known as Glässer’s disease. Several virulence factors have been described in H. parasuis including the virulence-associated trimeric autotransporters (VtaAs). VtaA2 is up-regulated during infection and is only found in virulent strains. In order to determine its biological function, the vtaA2 gene was cloned with its native promotor region in pACYC184, and the transformed Escherichia coli was used to perform functional in vitro assays. VtaA2 was found to have a role in attachment to plastic, mucin, BSA, fibronectin and collagen. As other VtaAs from H. parasuis, the passenger domain of VtaA2 contains collagen domains. In order to examine the contribution of the collagen repeats to VtaA2 function, a recombinant vtaA2 without the central collagen domains was obtained and named vtaA2OL. VtaA2OL showed similar capacity than VtaA2 to adhere to plastic, mucin, BSA, fibronectin and plasma but a reduced capacity to adhere to collagen, suggesting that the collagen domains of VtaA2 are involved in collagen attachment. No function in cell adhesion and invasion to epithelial alveolar cell line A549 or unspecific binding to primary alveolar macrophages was found. Likewise VtaA2 had no role in serum or phagocytosis resistance. We propose that VtaA2 mediates adherence to the host by binding to the mucin, found in the upper respiratory tract mucus, and to the extracellular matrix proteins, present in the connective tissue of systemic sites, such as the serosa

VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages

Haemophilus parasuis, a member of the family Pasteurellaceae, is a common inhabitant of the upper respiratory tract of healthy pigs and the etiological agent of Glässer's disease. As other virulent Pasteurellaceae, H. parasuis can prevent phagocytosis, but the bacterial factors involved in this virulence mechanism are not known. In order to identify genes involved in phagocytosis resistance, we constructed a genomic library of the highly virulent reference strain Nagasaki and clones were selected by increased survival after incubation with porcine alveolar macrophages (PAM). Two clones containing two virulent-associated trimeric autotransporter (VtaA) genes, vtaA8 and vtaA9, respectively, were selected by this method. A reduction in the interaction of the two clones with the macrophages was detected by flow cytometry. Monoclonal antibodies were produced and used to demonstrate the presence of these proteins on the bacterial surface of the corresponding clone, and on the H. parasuis phagocytosis-resistant strain PC4-6P. The effect of VtaA8 and VtaA9 in the trafficking of the bacteria through the endocytic pathway was examined by fluorescence microscopy and a delay was detected in the localization of the vtaA8 and vtaA9 clones in acidic compartments. These results are compatible with a partial inhibition of the routing of the bacteria via the degradative phagosome. Finally, antibodies against a common epitope in VtaA8 and VtaA9 were opsonic and promoted phagocytosis of the phagocytosis-resistant strain PC4-6P by PAM. Taken together, these results indicate that VtaA8 and VtaA9 are surface proteins that play a role in phagocytosis resistance of H. parasuis

Heterogeneity of Moraxella isolates found in the nasal cavities of piglets

Background Previous studies have shown that the genus Moraxella is commonly present in the nasal microbiota of swine. Results In this study, 51 isolates of Moraxella were obtained from nasal swabs from 3 to 4 week old piglets, which represented 26 different fingerprintings by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Whole 16S rRNA gene sequencing allowed the identification at species level of the Moraxella spp. isolates. The majority of the field strains were identified as Moraxella pluranimalium, but Moraxella porci was also detected. In addition, a cluster of 7 strains did not group with any described Moraxella species, probably representing a new species. Subsequent phenotypic characterization indicated that strains of Moraxella pluranimalium were mainly sensitive to serum complement, while the cluster representing the putative new species was highly resistant. Biofilm formation capacity was very variable among the Moraxella spp. isolates, while adherence to epithelial cell lines was similar among selected strains. Additionally, variability was also observed in the association of selected strains to porcine alveolar macrophages. Antimicrobial tests evidenced the existence of multidrug-resistance in the strains. Conclusions In summary, phenotypic characterization revealed heterogeneity among Moraxella strains from the nasal cavity of piglets. Strains with pathogenic potential were detected as well as those that may be commensal members of the nasal microbiota. However, the role of Moraxella in porcine diseases and health should be further evaluated.info:eu-repo/semantics/publishedVersio

Efficacy of Two Licensed Avian Influenza H5 Vaccines Against Challenge with a 2015 U.S. H5N2 clade 2.3.4.4 Highly Pathogenic Avian Influenza Virus in Domestic Ducks

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses from the H5 goose/Guangdong lineage caused a major outbreak in poultry in the United States in 2015. Although the outbreak was controlled, vaccines were considered as an alternative control method, and new vaccines were approved and purchased by the U.S. Department of Agriculture National Veterinary Stockpile for emergency use. In this study, we evaluated the efficacy of two of these vaccines in protecting Pekin ducks (Anas platyrhynchos var. domestica) against challenge with a H5N2 HPAI poultry isolate. A recombinant alphavirus-based vaccine and an inactivated adjuvanted reverse genetics vaccine, both expressing the hemagglutinin gene of a U.S. H5 clade 2.3.4.4 isolate (A/Gyrfalcon/Washington/41088-6/2014 H5N8), were used to immunize the ducks. The vaccines were given either as single vaccination at 2 days of age or in a prime-boost strategy at 2 and 15 days of age. At 32 days of age, all ducks were challenged with A/turkey/Minnesota/12582/15 H5N2 HPAI virus clade 2.3.4.4. All ducks from the nonvaccinated challenge control group became infected and shed virus; one duck in this group presented mild ataxia, and a second duck died. No mortality or clinical signs were observed in vaccinated and challenged ducks, with the exception of one duck presenting with mild ataxia. Both vaccines, regardless of the vaccination strategy used, were immunogenic in ducks and reduced or prevented virus shedding after challenge. In conclusion, good protection against H5Nx infection was achieved in ducks vaccinated with the vaccines examined, which were homologous to the challenge virus, with prime-boost strategies conferring the best protection against infection.info:eu-repo/semantics/publishedVersio

Insights on the interaction between Haemophilus parasuis and alveolar macrophages

Haemophilus parasuis és un bacteri de la família Pasteurellaceae. És un colonitzador del tracte respiratori superior en animals sans i l'agent etiològic de la malaltia de Glässer. Les soques de H. parasuis presenten diferències en virulència, que han estat observades tant en infeccions in vivo com en proves de fagocitosi in vitro amb macròfags alveolars porcins (PAMs). La patogenicitat de les soques ha estat correlacionada amb la resistència a la fagocitosi, però els factors de virulència implicats son desconeguts. Per la identificació dels factors implicats en la resistència a la fagocitosi, es va generar una llibreria genòmica de la soca virulenta de referència Nagasaki. De la incubació d'aquesta llibreria amb PAMs, es varen seleccionar dos clons amb gens codificant dos autotransportadors trimètrics associats a virulència (VtaA), vtaA8 i vtaA9. Mitjançant citometria de flux, es va aprofundir en el paper d'aquestes molècules en ambdós clons, els quals van mostrar una menor interacció amb els PAMs. La producció d'anticossos monoclonals (mAb) contra les proteïnes recombinants VtaA8 i VtaA9 van permetre determinar-ne la localització a la superfície dels clons. Els mateixos mAb detectaren aquestes proteïnes a la superfície de la soca resistent a la fagocitosi PC4-6P, però no a la soca avirulenta F9. Addicionalment, estudis amb microscòpia de fluorescència varen determinar l'efecte de VtaA8 i VtaA9 en el transport a la ruta endocítica, tot detectant un retard en la co-localització dels clons vtaA8 i vtaA9 amb compartiments àcids. Aquests resultats són compatibles amb una inhibició parcial del transport del bacteri a través de la degradació per fagosoma. Finalment, els anticossos contra un epítop comú a VtaA8 i VtaA9 van ser opsonitzadors i varen promoure la internalització de la soca resistent a la fagocitosi PC4-6 pels PAMs. Globalment, aquests resultats indiquen que VtaA8 i VtaA9 són proteïnes de superfície i juguen un paper en la resistència a la fagocitosi. La infecció de garrins privats de calostre nascuts de part natural amb soques de H. parasuis va mostrar diferències en el grau de virulència. Es varen emprar quatre soques de H. parasuis: les soques de referència virulenta Nagasaki i avirulenta SW114, la soca de camp IT29205 (obtinguda d'una lesió i virulenta en una infecció anterior) i la soca F9 (aïllada de la cavitat nasal d'un garrí sa). Els animals es varen inocular per via intranasal amb 107-108 CFU per individu. Dos animals no infectats s'utilitzaren com a controls. A diferents temps (1, 2, 4 i 7 dies post-infecció [dpi]), dos animals de cada grup es varen eutanasiar, i es varen prendre mostres de sèrum i del fluid bronquialveolar. Mitjançant citometria de flux, es varen analitzar els macròfags alveolars avaluant l'expressió dels marcadors de superfície CD163, CD172a, SLAI, SLAII, sialoadhesina, CD14 i SWC8. En funció de la virulència de la soca es varen poder observar canvis en el fenotip dels macròfags. A la fase inicial de la infecció (1 dpi), les soques no virulentes SW114 i F9 varen induir major expressió de CD163, sialoadhesina, SLAII i CD172a que les soques virulentes Nagasaki i IT29205. A 2 dpi, la situació canvià diametralment. Les soques virulentes generaren una forta inducció de l'expressió de CD172a, CD163 i sialoadhesina, seguida a continuació d'un sobtat increment d'IL-8 i CD163 soluble a 3-4 dpi. L'activació primerenca dels macròfags per part de les soques virulentes podria ser crítica per originar malaltia. L'associació entre el retard produït per les proteïnes VtaA8 i VtaA9 i la ruta endocítica, així com el retard en l'activació del macròfag, requereix estudis ulteriors.Haemophilus parasuis, a member of the family Pasteurellaceae, is a colonizer of the upper respiratory tract of healthy pigs and the etiological agent of Glässer's disease. Differences in virulence among H. parasuis strains have been widely observed by different tests, including in vivo infections and in vitro phagocytosis assays with porcine alveolar macrophages (PAMs). The pathogenicity of the strains has been correlated with resistance to phagocytosis, but the bacterial factors implicated are not known. To identify virulence factors involved in phagocytosis resistance, a genomic library of the virulent reference strain Nagasaki was produced and exposed to PAMs. After incubation with PAMs, two clones carrying two different virulent-associated trimeric autotransporters (VtaA), vtaA8 and vtaA9, were selected. The role of these molecules was further investigated and a reduction in the interaction of the two clones with the macrophages was detected by flow cytometry. Monoclonal antibodies (mAb) produced against the recombinant VtaA8 and VtaA9 proteins demonstrated the presence of these proteins on the bacterial surface of the corresponding clone. The same mAb also detected the proteins on the surface of H. parasuis phagocytosis-resistant strain PC4-6P, but not on the non-virulent strain F9. The effect of VtaA8 and VtaA9 in the trafficking of the bacteria through the endocytic pathway was examined by fluorescence microscopy and a delay was detected in the localization of the vtaA8 and vtaA9 clones in acidic compartments. Although VtaA8 and VtaA9 delayed phagocytosis, were not sufficient to completely inhibit the process. These results are compatible with a partial inhibition of the routing of the bacteria via the degradative phagosome. Finally, antibodies against a common epitope in VtaA8 and VtaA9 were opsonic and promoted phagocytosis of the phagocytosis-resistant strain PC4-6P by PAMs. Taken together, these results indicate that VtaA8 and VtaA9 are surface proteins that play a role in phagocytosis resistance of H. parasuis. Infection of snatch farrowed, colostrum-deprived piglets with different strains of H. parasuis demonstrated differences in the degree of virulence. We used four strains of H. parasuis: reference virulent strain Nagasaki, reference non-virulent strain SW114, and field strains IT29205 (from systemic lesion and virulent in a previous challenge) and F9 (from nasal cavity of a healthy piglet). The infection was performed intranasally with 107-108 CFU per animal. Two non-infected animals served as controls. At different times after infection (1, 2, 4 and 7 days post-infection [dpi]), two animals of each group were euthanized and bronchoalveolar lavages and sera were collected. Alveolar macrophages were analyzed for the expression of surface markers CD163, CD172a, SLAI, SLAII, sialoadhesin, CD14 and SWC8 by flow cytometry. The phenotype of macrophages changed along with the infection depending on the virulence of the strain. At early time-points (1 dpi), non-virulent strains SW114 and F9 induced higher expression of CD163, sialoadhesin, SLAII and CD172a than virulent strains Nagasaki and IT29205. At 2 dpi, the situation switched to a strong induction of expression of CD172a, CD163 and sialoadhesin by the virulent strains, which was followed by a steep increase in IL-8 and soluble CD163 at 3-4 dpi. The early delay in macrophage activation by virulent strains may be critical for disease establishment. The association between the delay produced by VtaA8 and VtaA9 in the endocytic route and the delay in macrophage activation needs further study

Differential interactions of virulent and non-virulent H. parasuis strains with naïve or swine influenza virus pre-infected dendritic cells

Pigs possess a microbiota in the upper respiratory tract that includes Haemophilus parasuis. Pigs are also considered the reservoir of influenza viruses and infection with this virus commonly results in increased impact of bacterial infections, including those by H. parasuis. However, the mechanisms involved in host innate responses towards H. parasuis and their implications in a co-infection with influenza virus are unknown. Therefore, the ability of a non-virulent H. parasuis serovar 3 (SW114) and a virulent serovar 5 (Nagasaki) strains to interact with porcine bone marrow dendritic cells (poBMDC) and their modulation in a co-infection with swine influenza virus (SwIV) H3N2 was examined. At 1 hour post infection (hpi), SW114 interaction with poBMDC was higher than that of Nagasaki, while at 8 hpi both strains showed similar levels of interaction. The co-infection with H3N2 SwIV and either SW114 or Nagasaki induced higher levels of IL-1β, TNF-α, IL-6, IL-12 and IL-10 compared to mock or H3N2 SwIV infection alone. Moreover, IL-12 and IFN-α secretion differentially increased in cells co-infected with H3N2 SwIV and Nagasaki. These results pave the way for understanding the differences in the interaction of non-virulent and virulent strains of H. parasuis with the swine immune system and their modulation in a viral co-infection

Fecal microbiota transplantation from warthog to pig confirms the influence of the gut microbiota on African swine fever susceptibility

African swine fever virus (ASFV) is the causative agent of a devastating hemorrhagic disease (ASF) that affects both domestic pigs and wild boars. Conversely, ASFV circulates in a subclinical manner in African wild pigs, including warthogs, the natural reservoir for ASFV. Together with genetic differences, other factors might be involved in the differential susceptibility to ASF observed among Eurasian suids (Sus scrofa) and African warthogs (Phacochoerus africanus). Preliminary evidence obtained in our laboratory and others, seems to confirm the effect that environmental factors might have on ASF infection. Thus, domestic pigs raised in specific pathogen-free (SPF) facilities were extremely susceptible to highly attenuated ASFV strains that were innocuous to genetically identical domestic pigs grown on conventional farms. Since gut microbiota plays important roles in maintaining intestinal homeostasis, regulating immune system maturation and the functionality of the innate/adaptive immune responses, we decided to examine whether warthog fecal microbiota transplantation (FMT) to domestic pigs affects host susceptibility to ASFV. The present work demonstrates that warthog FMT is not harmful for domestic weaned piglets, while it modifies their gut microbiota; and that FMT from warthogs to pigs confers partial protection against attenuated ASFV strains. Future work is needed to elucidate the protective mechanisms exerted by warthog FMT.info:eu-repo/semantics/publishedVersio

Fecal microbiota transplantation from warthog to pig confirms the influence of the gut microbiota on African swine fever susceptibility

African swine fever virus (ASFV) is the causative agent of a devastating hemorrhagic disease (ASF) that affects both domestic pigs and wild boars. Conversely, ASFV circulates in a subclinical manner in African wild pigs, including warthogs, the natural reservoir for ASFV. Together with genetic differences, other factors might be involved in the differential susceptibility to ASF observed among Eurasian suids (Sus scrofa) and African warthogs (Phacochoerus africanus). Preliminary evidence obtained in our laboratory and others, seems to confirm the effect that environmental factors might have on ASF infection. Thus, domestic pigs raised in specific pathogen-free (SPF) facilities were extremely susceptible to highly attenuated ASFV strains that were innocuous to genetically identical domestic pigs grown on conventional farms. Since gut microbiota plays important roles in maintaining intestinal homeostasis, regulating immune system maturation and the functionality of the innate/adaptive immune responses, we decided to examine whether warthog fecal microbiota transplantation (FMT) to domestic pigs affects host susceptibility to ASFV. The present work demonstrates that warthog FMT is not harmful for domestic weaned piglets, while it modifies their gut microbiota; and that FMT from warthogs to pigs confers partial protection against attenuated ASFV strains. Future work is needed to elucidate the protective mechanisms exerted by warthog FMT