Sow Vaccination with a Protein Fragment against Virulent Glaesserella (Haemophilus) parasuis Modulates Immunity Traits in Their Offspring.

Glaesserella (Haemophilus) parasuis, an early colonizer of the nasal cavity in piglets, is a highly heterogeneous species, comprising both commensal and virulent strains. Virulent G. parasuis strains can cause fibrinous polyserositis called Glässer's disease. Colostrum is a source of passive immunity for young piglets. When vaccinating sows, protective antibodies are transferred to their offspring through the colostrum. Here, sow vaccination was performed with a protein fragment, F4, from the outer membrane trimeric autotransporters VtaAs exclusively found in virulent G. parasuis. Piglets were allowed to suckle for 3 weeks, following which a challenge with two virulent strains of G. parasuis was performed. A group of nonvaccinated sows and their piglets were included as a control. Antibodies against F4 were confirmed using ELISA in the vaccinated sows and their offspring before the G. parasuis challenge. Compared to the control group, F4-vaccination also resulted in an increased level of serum TGF-ß both in vaccinated sows and in their offspring at early time points of life. After the challenge, a lower body temperature and a higher weight were observed in the group of piglets from vaccinated sows. One piglet from the non-vaccinated group succumbed to the infection, but no other significant differences in clinical signs were noticed. At necropsy, performed 2 weeks after the virulent challenge, the level of surfactant protein D (SP-D) in bronchoalveolar lavage was higher in the piglets from vaccinated sows. Vaccination did not inhibit the nasal colonization of the piglets by the challenge strains.

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.

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.

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.

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.

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.

Eficacia de dos vacunas con licencia contra influenza aviar H5 frente a un desafío con un virus de la influenza aviar altamente patógeno H5N2 en patos domésticos de los Estados Unidos del año 2015 y del clado 2015 2.3.4.4. Los virus de la influenza aviar altamente patógena (HPAI) 2.3.4.4 del linaje H5 ganso/Guangdong causaron un brote importante en la avicultura de los Estados Unidos en el año 2015. Aunque el brote fue controlado, las vacunas se consideraron un método de control alternativo y nuevas vacunas fueron aprobadas y adquiridas por la Reserva Nacional Veterinaria del Departamento de Agricultura de los Estados Unidos para uso en caso de emergencia. En este estudio, se evaluó la eficacia de dos de estas vacunas en la protección de patos Pekin frente al desafío con un aislamiento aviar H5N2 de alta patogenicidad. Se utilizaron una vacuna recombinante basada en alfavirus y una vacuna generada por genética inversa, inactivada y con adyuvante, ambas expresando el gene de la hemaglutinina de un aislamiento H5 clado 2.3.4.4 (A/Gyrfalcon/Washington/41088-6/2014 H5N8), para inmunizar los patos. Las vacunas se administraron como vacunación única a los 2 días de edad o con un programa de primovacunación y refuerzo a los 2 y 15 días de edad. A los 32 días de edad, todos los patos fueron desafiados con el virus de alta patogenicidad A/turkey/Minnesota/12582/15 H5N2 clado 2.3.4.4. Todos los patos del grupo control no vacunado y desafiado se infectaron y excretaron al virus; un pato en este grupo presentó ataxia leve y un segundo pato murió. No se observó mortalidad o signos clínicos en patos vacunados y desafiados, con la excepción de un pato que presentó ataxia leve. Ambas vacunas, independientemente de la estrategia de vacunación utilizada, fueron inmunogénicas en patos y redujeron o evitaron la diseminación del virus después del desafío. En conclusión, se logró una buena protección contra la infección por H5N2 en los patos vacunados con las vacunas evaluadas, las cuales eran homólogas al virus de desafío y las estrategias de primovacunación y refuerzo confirieron la mejor protección contra la infección.

Infectivity, transmission and pathogenicity of H5 highly pathogenic avian influenza clade 2.3.4.4 (H5N8 and H5N2) United States index viruses in Pekin ducks and Chinese geese.

In late 2014, a H5N8 highly pathogenic avian influenza (HPAI) virus, clade 2.3.4.4, spread by migratory waterfowl into North America reassorting with low pathogenicity AI viruses to produce a H5N2 HPAI virus. Since domestic waterfowl are common backyard poultry frequently in contact with wild waterfowl, the infectivity, transmissibility, and pathogenicity of the United States H5 HPAI index viruses (H5N8 and H5N2) was investigated in domestic ducks and geese. Ducks infected with the viruses had an increase in body temperature but no or mild clinical signs. Infected geese did not show increase in body temperature and most only had mild clinical signs; however, some geese presented severe neurological signs. Ducks became infected and transmitted the viruses to contacts when inoculated with high virus doses [(104 and 106 50% embryo infective dose (EID50)], but not with a lower dose (102 EID50). Geese inoculated with the H5N8 virus became infected regardless of the virus dose given, and transmitted the virus to direct contacts. Only geese inoculated with the higher doses of the H5N2 and their contacts became infected, indicating differences in infectivity between the two viruses and the two waterfowl species. Geese shed higher titers of virus and for a longer period of time than ducks. In conclusion, the H5 HPAI viruses can infect domestic waterfowl and easily transmit to contact birds, with geese being more susceptible to infection and disease than ducks. The disease is mostly asymptomatic, but infected birds shed virus for several days representing a risk to other poultry species.

Identification of a surface epitope specific of virulent strains of Haemophilus parasuis.

Haemophilus parasuis is a bacterium from the Pasteurellaceae family that comprises strains of different degree of virulence. Non-virulent strains are considered components of the upper respiratory tract microbiota, while virulent strains can invade systemic organs and cause fibrinous polyserositis (Glässer's disease). Genomic comparison of virulent and non-virulent strains led to the identification of a family of genes differentially associated to virulence, the virulence-associated trimeric autotransporters (vtaA). Monoclonal antibody 69C6 reacted with the surface of virulent strains and has allowed now the identification of an epitope in the C terminus of the passenger domain of the VtaAs from virulent strains. Protein modelling indicated that the epitope is probably exposed, although sera from pigs vaccinated with the passenger domain of VtaA9 and from convalescent animals did not react with the 69C6 epitope. Induction of antibodies against the 69C6 epitope by vaccination would allow a response targeting specifically virulent strains of H. parasuis.

Changes in adaptation of H5N2 highly pathogenic avian influenza H5 clade 2.3.4.4 viruses in chickens and mallards.

H5N2 highly pathogenic avian influenza (HPAI) viruses caused a severe poultry outbreak in the United States (U.S.) during 2015. In order to examine changes in adaptation of this viral lineage, the infectivity, pathogenicity and transmission of poultry H5N2 viruses were investigated in chickens and mallards in comparison to the wild duck 2014 U.S. index H5N2 virus. The four poultry isolates examined had a lower mean bird infectious dose than the index virus but still transmitted poorly to direct contacts. In mallards, two of the H5N2 poultry isolates had similar high infectivity and transmissibility as the index H5N2 virus, the H5N8 U.S. index virus, and a 2005 H5N1 clade 2.2 virus. Mortality occurred with the H5N1 virus and, interestingly, with one of two poultry H5N2 isolates. Increased virus adaptation to chickens was observed with the poultry H5N2 viruses; however these viruses retained high adaptation to mallards but pathogenicity was differently affected.

Pathogenicity and Transmission of H5 and H7 Highly Pathogenic Avian Influenza Viruses in Mallards.

Wild aquatic birds have been associated with the intercontinental spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the A/goose/Guangdong/1/96 (Gs/GD) lineage during 2005, 2010, and 2014, but dispersion by wild waterfowl has not been implicated with spread of other HPAI viruses. To better understand why Gs/GD H5 HPAI viruses infect and transmit more efficiently in waterfowl than other HPAI viruses, groups of mallard ducks were challenged with one of 14 different H5 and H7 HPAI viruses, including a Gs/GD lineage H5N1 (clade 2.2) virus from Mongolia, part of the 2005 dispersion, and the H5N8 and H5N2 index HPAI viruses (clade 2.3.4.4) from the United States, part of the 2014 dispersion. All virus-inoculated ducks and contact exposed ducks became infected and shed moderate to high titers of the viruses, with the exception that mallards were resistant to Ck/Pennsylvania/83 and Ck/Queretaro/95 H5N2 HPAI virus infection. Clinical signs were only observed in ducks challenged with the H5N1 2005 virus, which all died, and with the H5N8 and H5N2 2014 viruses, which had decreased weight gain and fever. These three viruses were also shed in higher titers by the ducks, which could facilitate virus transmission and spread. This study highlights the possible role of wild waterfowl in the spread of HPAI viruses. IMPORTANCE: The spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the Gs/GD lineage by migratory waterfowl is a serious concern for animal and public health. H5 and H7 HPAI viruses are considered to be adapted to gallinaceous species (chickens, turkeys, quail, etc.) and less likely to infect and transmit in wild ducks. In order to understand why this is different with certain Gs/GD lineage H5 HPAI viruses, we compared the pathogenicity and transmission of several H5 and H7 HPAI viruses from previous poultry outbreaks to Gs/GD lineage H5 viruses, including H5N1 (clade 2.2), H5N8 and H5N2 (clade 2.3.4.4) viruses, in mallards as a representative wild duck species. Surprisingly, most HPAI viruses examined in this study replicated well and transmitted among mallards; however, the three Gs/GD lineage H5 HPAI viruses replicated to higher titers, which could explain the transmission of these viruses in susceptible wild duck populations.

Effect of Infection with a Mesogenic Strain of Newcastle Disease Virus on Infection with Highly Pathogenic Avian Influenza Virus in Chickens.

Little is known on the interactions between avian influenza virus (AIV) and Newcastle disease virus (NDV) when coinfecting the same poultry host. In a previous study we found that infection of chickens with a mesogenic strain of NDV (mNDV) can reduce highly pathogenic AIV (HPAIV) replication, clinical disease, and mortality. This interaction depended on the titer of the viruses used and the timing of the infections. To further explore the effect of mNDV infectious dose in protecting chickens against HPAIV infection, 2-wk-old birds were inoculated with different doses of mNDV (10(4), 10(6), or 10(7) 50% embryo infective dose [EID50]) 3 days before inoculation with a HPAIV (10(5) or 10(6) EID50). Although birds coinfected with the higher mNDV doses (10(6) or 10(7)) survived for longer than birds inoculated only with HPAIV (10(5)), we did not observe the same protection with the lower dose of mNDV (10(4)) or when given the higher dose of HPAIV (10(6)), indicating that the relation between the titer of the two coinfecting viruses is determinant in the outcome. In a similar experiment, a higher number of 4-wk-old birds survived, and for longer, even when given higher HPAIV doses (10(6.3) and 10(7.3) EID50). In addition, we also examined the duration of protection provided by mNDV (10(7) EID50) on a HPAIV infection. Five-week-old chickens were inoculated with mNDV followed by inoculation with 10(6) EID50 of an HPAIV given at 2, 4, 6, or 9 days after the mNDV. HPAIV replication was affected and an increase in survival was found in all coinfected groups when compared to the HPAIV single-inoculated group, but the mortality in coinfected groups was high. In conclusion, previous inoculation with mNDV can affect HPAIV replication in chickens for at least 9 days, but this viral interference is titer dependent.

Efficacy of a Recombinant Turkey Herpesvirus H5 Vaccine Against Challenge With H5N1 Clades 1.1.2 and 2.3.2.1 Highly Pathogenic Avian Influenza Viruses in Domestic Ducks (Anas platyrhynchos domesticus).

Domestic ducks are the second most abundant poultry species in many Asian countries and have played a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI).In this study, the protective efficacy of a live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 HPAI strain (A/Swan/Hungary/4999/ 2006) (rHVT-H5/2.2), given at 3 days of age, was examined in Pekin ducks (Anas platyrhynchos domesticus). The vaccine was given alone or in combination with an inactivated H5N1 clade 2.3.2.1 reverse genetic (rgGD/2.3.2.1) vaccine given at 16 days of age, either as a single vaccination or in a prime-boost regime. At 30 days of age, ducks were challenged with one of two H5N1 HPAI viruses: A/duck/Vietnam/NCVD-2721/2013 (clade 1.1.2) or A/duck/Vietnam/NCVD-1584/2012 (clade 2.3.2.1.C). These viruses produced 100% mortality in less than 5 days in nonvaccinated control ducks. Ducks vaccinated with the rgGD/2.3.2.1 vaccine, with or without the rHVT-H5/2.2 vaccine, were 90%-100% protected against mortality after challenge with either of the two H5N1 HPAI viruses. The rHVT-H5/2.2 vaccine alone, however, conferred only 30% protection against mortality after challenge with either H5N1 HPAI virus; the surviving ducks from these groups shed higher amount of virus and for longer than the single-vaccinated rgGD/2.3.2.1 group. Despite low protection, ducks vaccinated with the rHVT-H5/2.2 vaccine and challenged with the clade 1.1.2 Vietnam virus had a longer mean death time than nonvaccinated controls (P = 0.02). A booster effect was found on reduction of virus shedding when using both vaccines, with lower oropharyngeal viral titers at 4 days after challenge with either HPAI virus (P < 0.05). Neither rHVT-H5/2.2 nor standard HVT vaccine could be detected in samples collected from multiple tissues at different time points, indicting minimal levels of viral replication. In conclusion, although a minor effect on survival was observed, this study demonstrates the suboptimal protection with the rHVT-H5/2.2 vaccine given alone in Pekin ducks against H5N1 HPAI viruses and only a minor additive effect on virus shedding reduction when used with an inactivated vaccine in a prime-boost regime.

Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication, disease, and mortality in chickens.

Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (10(6.9) EID50) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (10(5.3-5.5) EID50) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.

Experimental co-infections of domestic ducks with a virulent Newcastle disease virus and low or highly pathogenic avian influenza viruses.

Infections with avian influenza viruses (AIV) of low and high pathogenicity (LP and HP) and Newcastle disease virus (NDV) are commonly reported in domestic ducks in many parts of the world. However, it is not clear if co-infections with these viruses affect the severity of the diseases they produce, the amount of virus shed, and transmission of the viruses. In this study we infected domestic ducks with a virulent NDV virus (vNDV) and either a LPAIV or a HPAIV by giving the viruses individually, simultaneously, or sequentially two days apart. No clinical signs were observed in ducks infected or co-infected with vNDV and LPAIV, but co-infection decreased the number of ducks shedding vNDV and the amount of virus shed (P<0.01) at 4 days post inoculation (dpi). Co-infection did not affect the number of birds shedding LPAIV, but more LPAIV was shed at 2 dpi (P<0.0001) from ducks inoculated with only LPAIV compared to ducks co-infected with vNDV. Ducks that received the HPAIV with the vNDV simultaneously survived fewer days (P<0.05) compared to the ducks that received the vNDV two days before the HPAIV. Co-infection also reduced transmission of vNDV to naïve contact ducks housed with the inoculated ducks. In conclusion, domestic ducks can become co-infected with vNDV and LPAIV with no effect on clinical signs but with reduction of virus shedding and transmission. These findings indicate that infection with one virus can interfere with replication of another, modifying the pathogenesis and transmission of the viruses.

Time course Haemophilus parasuis infection reveals pathological differences between virulent and non-virulent strains in the respiratory tract.

Haemophilus parasuis is a common inhabitant of the upper respiratory tract of pigs and the etiological agent of Glässer's disease. However, the host-pathogen interaction remains to be well understood. In this work, 33 colostrum-deprived pigs were divided in 4 groups and each group was inoculated intranasally with a different H. parasuis strain (non-virulent strains SW114 and F9, and virulent strains Nagasaki and IT29755). Animals were necropsied at different times in order to determine the location of the bacteria in the respiratory tract of the host during infection. An immunohistochemistry method was developed to detect H. parasuis in nasal turbinates, trachea and lung. Also, the co-localization of H. parasuis with macrophages or neutrophils was examined by double immunohistochemistry and double immunofluorescence. Virulent strains showed a biofilm-like growth in nasal turbinates and trachea and were found easily in lung. Some virulent bacteria were detected in association with macrophages and neutrophils, but also inside pneumocyte-like cells. On the other hand, non-virulent strains were seldom detected in nasal turbinates and trachea, where they showed a microcolony pattern. Non-virulent strains were essentially not detected in lung. In conclusion, this work presents data showing differential localization of H. parasuis bacteria depending on their virulence. Interestingly, the intracellular location of virulent H. parasuis bacteria in non-phagocytic cells in lung could allow the persistence of the bacteria and constitute a virulence mechanism.

Role of poultry in the spread of novel H7N9 influenza virus in China.

UNLABELLED: The recent outbreak of H7N9 influenza in China has resulted in many human cases with a high fatality rate. Poultry are the likely source of infection for humans on the basis of sequence analysis and virus isolations from live bird markets, but it is not clear which species of birds are most likely to be infected and shedding levels of virus sufficient to infect humans. Intranasal inoculation of chickens, Japanese quail, pigeons, Pekin ducks, Mallard ducks, Muscovy ducks, and Embden geese with 10(6) 50% egg infective doses of the A/Anhui/1/2013 virus resulted in infection but no clinical disease signs. Virus shedding was much higher and prolonged in quail and chickens than in the other species. Quail effectively transmitted the virus to direct contacts, but pigeons and Pekin ducks did not. In all species, virus was detected at much higher titers from oropharyngeal swabs than cloacal swabs. The hemagglutinin gene from samples collected from selected experimentally infected birds was sequenced, and three amino acid differences were commonly observed when the sequence was compared to the sequence of A/Anhui/1/2013: N123D, N149D, and L217Q. Leucine at position 217 is highly conserved for human isolates and is associated with α2,6-sialic acid binding. Different amino acid combinations were observed, suggesting that the inoculum had viral subpopulations that were selected after passage in birds. These experimental studies corroborate the finding that certain poultry species are reservoirs of the H7N9 influenza virus and that the virus is highly tropic for the upper respiratory tract, so testing of bird species should preferentially be conducted with oropharyngeal swabs for the best sensitivity. IMPORTANCE: The recent outbreak of H7N9 influenza in China has resulted in a number of human infections with a high case fatality rate. The source of the viral outbreak is suspected to be poultry, but definitive data on the source of the infection are not available. This study provides experimental data to show that quail and chickens are susceptible to infection, shed large amounts of virus, and are likely important in the spread of the virus to humans. Other poultry species can be infected and shed virus but are less likely to play a role of transmitting the virus to humans. Pigeons were previously suggested to be a possible source of the virus because of isolation of the virus from several pigeons in poultry markets in China, but experimental studies show that they are generally resistant to infection and are unlikely to play a role in the spread of the virus.

Virus interference between H7N2 low pathogenic avian influenza virus and lentogenic Newcastle disease virus in experimental co-infections in chickens and turkeys.

Low pathogenicity avian influenza virus (LPAIV) and lentogenic Newcastle disease virus (lNDV) are commonly reported causes of respiratory disease in poultry worldwide with similar clinical and pathobiological presentation. Co-infections do occur but are not easily detected, and the impact of co-infections on pathobiology is unknown. In this study chickens and turkeys were infected with a lNDV vaccine strain (LaSota) and a H7N2 LPAIV (A/turkey/VA/SEP-67/2002) simultaneously or sequentially three days apart. No clinical signs were observed in chickens co-infected with the lNDV and LPAIV or in chickens infected with the viruses individually. However, the pattern of virus shed was different with co-infected chickens, which excreted lower titers of lNDV and LPAIV at 2 and 3 days post inoculation (dpi) and higher titers at subsequent time points. All turkeys inoculated with the LPAIV, whether or not they were exposed to lNDV, presented mild clinical signs. Co-infection effects were more pronounced in turkeys than in chickens with reduction in the number of birds shedding virus and in virus titers, especially when LPAIV was followed by lNDV. In conclusion, co-infection of chickens or turkeys with lNDV and LPAIV affected the replication dynamics of these viruses but did not affect clinical signs. The effect on virus replication was different depending on the species and on the time of infection. These results suggest that infection with a heterologous virus may result in temporary competition for cell receptors or competent cells for replication, most likely interferon-mediated, which decreases with time.

Advances in the quest for virulence factors of Haemophilus parasuis.

Haemophilus parasuis colonises healthy pigs and is the aetiological agent of Glässer's disease. The pathogenicity of H. parasuis is poorly characterised, while prevention and control of Glässer's disease continues to be challenging. Understanding the pathogenicity of H. parasuis is essential for determining how this bacterium produces disease and to better distinguish between virulent and non-virulent strains. Infection by H. parasuis requires adhesion to and invasion of host cells, resistance to phagocytosis by macrophages, resistance to serum complement and induction of inflammation. Identification of virulence factors involved in these mechanisms has been limited by difficulties in producing mutants in H. parasuis. Recent advances in understanding the pathogenesis of H. parasuis are due in part to the production of deletion mutants, although most of the potential virulence factors described so far require further characterisation. Data supporting the role of lipooligosaccharide, capsule formation, porin proteins, cytolethal distending toxin and trimeric autotransporters (VtaA), among other molecules, in the virulence of H. parasuis have been described. This review provides an overview of the current knowledge of virulence factors of H. parasuis.

Changes in macrophage phenotype after infection of pigs with Haemophilus parasuis strains with different levels of virulence.

Haemophilus parasuis is a colonizer of healthy piglets and the etiological agent of Glässer's disease. Differences in virulence among strains of H. parasuis have been widely observed. In order to explore the host-pathogen interaction, snatch-farrowed colostrum-deprived piglets were intranasally infected with 4 strains of H. parasuis: reference virulent strain Nagasaki, reference nonvirulent strain SW114, field strain IT29205 (from a systemic lesion and virulent in a previous challenge), and field strain F9 (from the nasal cavity of a healthy piglet). At different times after infection, two animals of each group were euthanized and alveolar macrophages were analyzed for the expression of CD163, CD172a, SLA I (swine histocompatibility leukocyte antigen I), SLA II, sialoadhesin (or CD169), and CD14. At 1 day postinfection (dpi), virulent strains induced reduced expression of CD163, SLA II, and CD172a on the surfaces of the macrophages, while nonvirulent strains induced increased expression of CD163, both compared to noninfected controls. At 2 dpi, the pattern switched into a strong expression of CD172a, CD163, and sialoadhesin by the virulent strains, which was followed by a steep increase in interleukin 8 (IL-8) and soluble CD163 in serum at 3 to 4 dpi. The early increase in surface expression of CD163 induced by nonvirulent strains went along with higher levels of IL-8 in serum than those induced by virulent strains in the first 2 days of infection. Alpha interferon (IFN-α) induction was observed only in animals infected with nonvirulent strains. Overall, these results are compatible with a delay in macrophage activation by virulent strains, which may be critical for disease production.

Serum cross-reaction among virulence-associated trimeric autotransporters (VtaA) of Haemophilus parasuis.

Glässer's disease is a fibrinous polyserositis and polyarthritis of swine caused by the bacterium Haemophilus parasuis. Control by vaccination has been limited for years due to lack of cross-protection among strains. However, 6 trimeric autotransporters (VtaA) of the Nagasaki strain were shown to be antigenic and gave partial protection to a lethal challenge. The antigenic relationship among the VtaAs was examined by immunizing mice with individual VtaA showing that they cross-reacted by ELISA mainly with VtaA from the same group. When sera from protected and non-protected vaccinated piglets were examined no differences in VtaA cross-reactivity profiles were found. In addition, sera from commercial pigs immunized with a single VtaA (VtaA9) showed a wider range of VtaA cross-reaction, probably due to the previous colonization by H. parasuis. These results can help the development of new vaccine formulations against H. parasuis by allowing a rational VtaA selection.