Campylobacter hepaticus in the Production Environment and Stagnant Water as a Potential Source of C. hepaticus Causing Spotty Liver Disease in Free-Range Laying Hens in Georgia, United States.

Spotty liver disease (SLD) has emerged as an important cause of disease in egg-producing flocks in countries such as the United Kingdom and Australia and has emerged in the United States. The organisms implicated in SLD include Campylobacter hepaticus and, more recently, Campylobacter bilis. These organisms have been found to cause focal lesions on the livers of infected birds. Campylobacter hepaticus infection results in reduced egg production, decreased feed consumption resulting in reduced egg size, and increased mortality of highly valuable hens. In the fall of 2021, birds from two flocks (A and B) of organic pasture-raised laying hens were submitted to the Poultry Diagnostic Research Center at the University of Georgia with a history suspicious of SLD. Postmortem examination of Flock A found 5/6 hens had small multifocal lesions on the liver and were PCR positive for C. hepaticus from pooled swab analysis of samples of the liver and gall bladder. Necropsy of Flock B found 6/7 submitted birds had spotty liver lesions. In pooled bile swabs, 2/7 hens from Flock B were also PCR positive for C. hepaticus. A follow-up visit to Flock A was scheduled 5 days later, as well as a visit to a flock where SLD has not been reported (Flock C), which was used as a comparative control. Samples of the liver, spleen, cecal tonsil, ceca, blood, and gall bladder were collected from six hens per house. Additionally, feed, water nipples, and environmental water (stagnant water outside the house) were collected from the affected farm and the control farm. To detect the organism, all samples collected were subjected to direct plating on blood agar and enrichment in Preston broth with incubation under microaerophilic conditions. After multiple phases of bacterial culture purification from all samples, single bacterial cultures displaying characteristics of C. hepaticus were tested by PCR to confirm identity. From Flock A, liver, ceca, cecal tonsils, gall bladder, and environmental water were PCR positive for C. hepaticus. No positive samples were detected in Flock C. After another follow-up visit, 10 wk later, Flock A was PCR positive for C. hepaticus from gall bladder bile and feces and one environmental water sample displayed a weak positive reaction for C. hepaticus. Flock C was PCR negative for C. hepaticus. To gain more knowledge about C. hepaticus prevalence, a survey of 6 layer hens from 12 different layer hen flocks between the ages of 7 to 80 wk, raised in different housing systems, were tested for C. hepaticus. The 12 layer hen flocks were culture and PCR negative for C. hepaticus. Currently, there are no approved treatments for C. hepaticus and no vaccine is available. The results of this study suggest that C. hepaticus may be endemic in some areas of the United States, and free-range laying hens may be exposed from the environment/stagnant water in areas where they range.

Campylobacter hepaticus en el ambiente de producción avícola y en el agua estancada como fuente potencial de C. hepaticus que causante de la necrosis hepática focal en gallinas ponedoras de corral en Georgia, Estados Unidos. La necrosis hepática focal (SLD, por sus siglas en inglés) se ha convertido en una causa importante de enfermedad en las parvadas productoras de huevo en países como el Reino Unido y Australia y también ha surgido en los Estados Unidos. Los organismos implicados en necrosis hepática focal incluyen Campylobacter hepaticus y, más recientemente, Campylobacter bilis. Se ha encontrado que estos organismos causan lesiones focales en el hígado de las aves infectadas. La infección por C. hepaticus da como resultado una reducción en la producción de huevos, una disminución en el consumo de alimento, lo que resulta en una reducción del tamaño de los huevos y una mayor mortalidad de gallinas de alto valor económico. En el otoño del 2021, aves de dos lotes (A y B) de gallinas de postura criadas en pastos orgánicos se enviaron al Centro de Diagnóstico e Investigación Avícolas de la Universidad de Georgia con antecedentes sospechosos de necrosis hepática focal. En el examen post mortem de la parvada A se encontró que cinco de un total de seis gallinas tenían pequeñas lesiones multifocales en el hígado y fueron positivas mediante PCR para C. hepaticus a partir de un análisis de hisopos combinados de muestras del hígado y de la vesícula biliar. La necropsia de la parvada B encontró que seis de un total de siete aves enviadas tenían lesiones hepáticas irregulares. En muestras agrupadas de bilis, dos de un total de siete gallinas de la parvada B también fueron positivas a C. hepaticus por PCR. Se programó una visita de seguimiento a la Parvada A cinco días después, así como una visita a una parvada en la que no se había reportado la presencia de necrosis hepática focal (Parvada C), que se utilizó como control para propósitos de comparación. Se recolectaron muestras de hígado, bazo, tonsilas cecales, sacos ciegos, sangre y vesícula biliar de seis gallinas por gallinero. Además, se recolectó alimento, muestras de agua de bebederos de niple y agua ambiental (agua estancada fuera de la casa) de la granja afectada y la granja de control. Para detectar el organismo, todas las muestras recolectadas se sometieron a siembra directa en agar sangre y enriquecimiento en caldo Preston con incubación en condiciones microaerófilas. Después de varias fases de purificación del cultivo bacteriano de todas las muestras, se analizaron mediante PCR los cultivos bacterianos individuales que mostraban características de C. hepaticus para confirmar la identidad. De la parvada A, el hígado, el ciego, las tonsilas cecales, la vesícula biliar y el agua ambiental dieron positivo por PCR para C. hepaticus. No se detectaron muestras positivas en la parvada C. Después una segunda visita de seguimiento, 10 semanas después, la parvada A mostró resultado positivo por PCR para C. hepaticus en la bilis de la vesícula biliar y en las heces, y una muestra de agua ambiental mostró una reacción positiva débil para C. hepaticus . La parvada C resultó negativa mediante PCR para C. hepaticus. Para obtener más conocimiento sobre la prevalencia de C. hepaticus, se realizó un muestreo incluyendo seis gallinas de postura de 12 lotes diferentes de gallinas ponedoras entre las edades de 7 a 80 semanas, criadas en diferentes sistemas de alojamiento, para detectar C. hepaticus. Las doce parvadas de gallinas de postura fueron negativas por cultivo y mediante PCR para C. hepaticus. Actualmente, no hay tratamientos aprobados para C. hepaticus y no hay vacuna disponible. Los resultados de este estudio sugieren que C. hepaticus puede ser endémico en algunas áreas de los Estados Unidos, y las gallinas de postura bajo pastoreo pueden estar expuestas al medio ambiente o al agua estancada en las áreas donde están alojadas.

Erysipelothrix rhusiopathiae Infection Associated with Swollen Head Syndrome in a Broiler Breeder Flock in North Georgia.

An unusual case of swollen head syndrome in a 55-wk-old broiler breeder flock was identified in north Georgia in the summer of 2019. The presenting complaint was elevated mortality and visibly swollen heads. Necropsy of affected birds on the farm primarily revealed signs of bacterial septicemia, with few large scab lesions near the vent area. Bacterial culture analysis identified multiple organisms, but the primary organism of interest was identified as Erysipelothrix rhusiopathiae, cultured from affected liver, lung, sinuses, and one swollen wattle of birds in the affected house. Histopathologic analysis identified gram-positive rod-shaped bacteria in the spleen and liver (consistent with bacterial septicemia) confirmed with special staining (Brown & Hopps Gram stain). These organisms were noted to be consistent with E. rhusiopathiae; E. rhusiopathiae infection in broiler breeder chickens is a rare occurrence and is primarily associated with turkeys and/or swine production systems.

Reporte de caso- Infección por Erysipelothrix rhusiopathiae asociada con el síndrome de cabeza hinchada en un pollo de engorde. En el verano del 2019, se identificó un caso inusual de síndrome de cabeza hinchada en una parvada de reproductoras de pollos de engorde de 55 semanas de edad en el norte de Georgia. Las observaciones iniciales incluyeron mortalidad elevada y cabezas visiblemente inflamadas. La necropsia de las aves afectadas en la granja reveló principalmente signos de septicemia bacteriana, con pocas lesiones costrosas grandes cerca del área de extractores de la ventilación. El análisis de cultivos bacterianos identificó múltiples organismos, pero el principal organismo de interés fue identificado como Erysipelothrix rhusiopathiae, cultivado a partir de hígado, pulmón, senos paranasales y de una barbilla inflamada de aves en la caseta afectada. El análisis histopatológico identificó bacterias grampositivas en forma de bastón en el bazo y el hígado (compatibles con septicemia bacteriana) confirmadas con tinción especial (tinción de Gram de Brown y Hopps). Se observó que estos organismos eran compatibles con E. rhusiopathiae; La infección por E. rhusiopathiae en pollos de engorde se presenta de forma esporádica y se asocia principalmente con sistemas de producción de pavos y/o cerdos.

Hemorrhagic Hepatopathy in Broiler Breeder Pullets: Gross and Microscopic Pathology and Factors Associated with Incidence.

Hemorrhagic hepatopathy is a syndrome reported in layer pullets resulting in mortality and lesions including hepatic, splenic, and intestinal necrosis; hepatic and splenic enlargement; hemorrhages; amyloidosis of the muscle, spleen, and liver; accumulation of noncoagulated hemorrhagic fluid in the coelom; and frequently, granulomatous myositis at bacterin injection sites. The syndrome is characterized in the literature in table egg layer pullets and is thought to be associated with the administration of bacterin vaccines, namely, frequently Salmonella enterica subsp. enterica bacterins. Hemorrhagic hepatopathy is recognized by industry veterinarians as also occurring infrequently in broiler breeder pullets in the United States. As the condition is likely due to an inflammatory process in response to bacterial lipopolysaccharide inoculation, it is important to characterize both the pathologic changes and predisposing factors for the condition in broiler breeds, which are immunologically different from table egg layer breeds. In this study, we characterize the gross and microscopic lesions observed in a series of diagnostic laboratory cases of hemorrhagic hepatopathy in broiler breeder pullets and suggest a possible pathophysiology for the condition. Additionally, we report results from a case survey of the United States broiler industry that suggest that the condition is due to a reaction to bacterin vaccination and that certain bacterin products may predispose pullet flocks to develop the condition. Although further research is indicated, these findings establish hemorrhagic hepatopathy as a pathologic condition of broiler breeder pullets and may aid in the diagnosis and prevention of the syndrome.

Artículo regular­La hepatopatía hemorrágica en pollitas reproductoras pesadas: Patología macroscópica y microscópica y factores asociados con la incidencia La hepatopatía hemorrágica es un síndrome reportado en pollitas ponedoras que resulta en mortalidad y lesiones, incluyendo necrosis hepática, esplénica e intestinal; agrandamiento hepático y esplénico; hemorragias; amiloidosis del músculo, bazo e hígado; acumulación de líquido hemorrágico no coagulado en la cavidad celómica; y con frecuencia, miositis granulomatosa en los lugares de inyección de bacterina. El síndrome se ha caracterizado en la bibliografía en pollitas ponedoras de huevo comercial y se cree que está asociado con la administración de vacunas de bacterianas, con frecuencia bacterinas de Salmonella. Los veterinarios de la industria reconocen que la hepatopatía hemorrágica también ocurre con poca frecuencia en pollitas de reproductoras pesadas en los Estados Unidos. Como es probable que esta condición se deba a un proceso inflamatorio en respuesta a la inoculación de lipopolisacáridos bacterianos, es importante caracterizar tanto los cambios patológicos como los factores predisponentes para la afección en las líneas de pollos de engorde, que son inmunológicamente diferentes de las líneas ponedoras de huevo comercial. En este estudio, se caracterizaron las lesiones macroscópicas y microscópicas observadas en una serie de casos de laboratorio de diagnóstico de hepatopatía hemorrágica en pollitas reproductoras de pollos de engorde y sugerimos una posible fisiopatología de esta condición. Además, se reportan los resultados de una encuesta de casos de la industria de pollos de engorde en los Estados Unidos que sugiere que la condición se debe a una reacción a la vacunación con bacterinas y que ciertos productos de las bacterinas pueden predisponer a las parvadas de pollitas a desarrollar la afección. Aunque se requieren más investigaciones, estos hallazgos establecen la hepatopatía hemorrágica como una condición patológica de las pollitas reproductoras pesadas y pueden ayudar en el diagnóstico y a la prevención del síndrome.

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.

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.

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.

Suboptimal protection against H5N1 highly pathogenic avian influenza viruses from Vietnam in ducks vaccinated with commercial poultry vaccines.

Domestic ducks are the second most abundant poultry species in many Asian countries including Vietnam, and play a critical role in the epizootiology of H5N1 highly pathogenic avian influenza (HPAI) [FAO]. In this study, we examined the protective efficacy in ducks of two commercial H5N1 vaccines widely used in Vietnam; Re-1 containing A/goose/Guangdong/1/1996 hemagglutinin (HA) clade 0 antigens, and Re-5 containing A/duck/Anhui/1/2006 HA clade 2.3.4 antigens. Ducks received two doses of either vaccine at 7 and at 14 or 21 days of age followed by challenge at 30 days of age with viruses belonging to the HA clades 1.1, 2.3.4.3, 2.3.2.1.A and 2.3.2.1.B isolated between 2008 and 2011 in Vietnam. Ducks vaccinated with the Re-1 vaccine were protected after infection with the two H5N1 HPAI viruses isolated in 2008 (HA clades 1.1 and 2.3.4.3) showing no mortality and limited virus shedding. The Re-1 and Re-5 vaccines conferred 90-100% protection against mortality after challenge with the 2010 H5N1 HPAI viruses (HA clade 2.3.2.1.A); but vaccinated ducks shed virus for more than 7 days after challenge. Similarly, the Re-1 and Re-5 vaccines only showed partial protection against the 2011 H5N1 HPAI viruses (HA clade 2.3.2.1.A and 2.3.2.1.B), with a high proportion of vaccinated ducks shedding virus for more than 10 days. Furthermore, 50% mortality was observed in ducks vaccinated with Re-1 and challenged with the 2.3.2.1.B virus. The HA proteins of the 2011 challenge viruses had the greatest number of amino acid differences from the two vaccines as compared to the viruses from 2008 and 2009, which correlates with the lesser protection observed with these viruses. These studies demonstrate the suboptimal protection conferred by the Re-1 and Re-5 commercial vaccines in ducks against H5N1 HPAI clade 2.3.2.1 viruses, and underscore the importance of monitoring vaccine efficacy in the control of H5N1 HPAI in ducks.

Effect of species, breed and route of virus inoculation on the pathogenicity of H5N1 highly pathogenic influenza (HPAI) viruses in domestic ducks.

H5N1 highly pathogenic avian influenza (HPAI) viruses continue to be a threat to poultry in many regions of the world. Domestic ducks have been recognized as one of the primary factors in the spread of H5N1 HPAI. In this study we examined the pathogenicity of H5N1 HPAI viruses in different species and breeds of domestic ducks and the effect of route of virus inoculation on the outcome of infection. We determined that the pathogenicity of H5N1 HPAI viruses varies between the two common farmed duck species, with Muscovy ducks (Cairina moschata) presenting more severe disease than various breeds of Anas platyrhynchos var. domestica ducks including Pekin, Mallard-type, Black Runners, Rouen, and Khaki Campbell ducks. We also found that Pekin and Muscovy ducks inoculated with two H5N1 HPAI viruses of different virulence, given by any one of three routes (intranasal, intracloacal, or intraocular), became infected with the viruses. Regardless of the route of inoculation, the outcome of infection was similar for each species but depended on the virulence of the virus used. Muscovy ducks showed more severe clinical signs and higher mortality than the Pekin ducks. In conclusion, domestic ducks are susceptible to H5N1 HPAI virus infection by different routes of exposure, but the presentation of the disease varied by virus strain and duck species. This information helps support the planning and implementation of H5N1 HPAI surveillance and control measures in countries with large domestic duck populations.

Differences in pathogenicity, response to vaccination, and innate immune responses in different types of ducks infected with a virulent H5N1 highly pathogenic avian influenza virus from Vietnam.

In a previous study, we found clear differences in pathogenicity and response to vaccination against H5N1 highly pathogenic avian influenza (HPAI; HA dade 2.3.4) between Pekin (Anas platyrhynchos var. domestica) and Muscovy (Cairina moschata) ducks vaccinated using a commercial inactivated vaccine (Re-1). The objective of the present study was to further investigate the pathogenicity of H5N1 HPAI viruses in different species of ducks by examining clinical signs and innate immune responses to infection with a different strain of H5N1 HPAI virus (HA clade 1) in two domestic ducks, Pekin and Muscovy, and one wild-type duck, mallard (Anas platyrhynchos). Protection conferred by vaccination using the Re-1 vaccine against infection with this virus was also compared between Pekin and Muscovy ducks. Differences in pathogenicity were observed among the virus-infected ducks, as the Muscovy ducks died 2 days earlier than did the Pekin and mallard ducks, and they presented more-severe neurologic signs. Conversely, the Pekin and mallard ducks had significantly higher body temperatures at 2 days postinfection (dpi) than did the Muscovy ducks, indicating possible differences in innate immune responses. However, similar expression of innate immune-related genes was found in the spleens of virus-infected ducks at this time point. In all three duck species, there was up-regulation of IFN-alpha, IFN-gamma, IL-6, CCL19, RIG-I, and MHC class I and down-regulation of MHC class II, but variable expression of IL-18 and TLR7. As in our previous study, vaccinated Muscovy ducks showed less protection against virus infection than did Pekin ducks, as evidenced by the higher mortality and higher number of Muscovy ducks shedding virus when compared to Pekin ducks. In conclusion, infection with an H5N1 HPAI virus produced a systemic infection with high mortality in all three duck species; however, the disease was more severe in Muscovy ducks, which also had a poor response to vaccination. The differences in response to virus infection could not be explained by differences in the innate immune responses between the different types of ducks when examined at 2 days dpi, and earlier time points need to be evaluated.

Effect of age on the pathogenesis and innate immune responses in Pekin ducks infected with different H5N1 highly pathogenic avian influenza viruses.

The pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in domestic ducks varies between different viruses and is affected by the age of the ducks, with younger ducks presenting a more severe disease. In order to better understand the pathobiology of H5N1 HPAI in ducks including the role of host responses, 2 and 5-week-old Pekin ducks were infected with three different H5N1 HPAI viruses. Virus-induced pathology ranged from no clinical signs to severe disease and mortality, with the 2-week-old ducks being more severely affected by the more virulent viruses. However, these more virulent viruses induced higher body temperatures in the 5-week-old ducks than in the 2-week-old ducks indicating possible differences in innate immune responses. To analyze the ducks host responses to H5N1 HPAI virus infection, expression of innate immune-related genes was measured in the spleens and lungs of infected ducks at the peak of virus infection. IFN-α, RIG-I, and IL-6 RNA levels were increased in spleens regardless of the virus given and the age of the ducks, however differences were observed in the levels of up-regulation of IFN-α and RIG-I between the 2 and the 5-week-old ducks with the more virulent virus. Differences in IL-2 gene expression were also observed. In the lungs, the levels of expression of innate immune-related genes were lower than in the spleen, with mostly up-regulation of RIG-I and IL-6 and down-regulation of IFN-α and IL-2; no significant difference in expression was found between the 2 and the 5-week-old ducks. The differences observed in the innate immune responses to infection with H5N1 HPAI viruses could explain in part the differences in pathogenicity found between the 2 and 5-week-old ducks, however earlier time points after infection and additional innate immune-related genes should be examined.

Susceptibility of avian species to North American H13 low pathogenic avian influenza viruses.

Gulls are widely recognized reservoirs for low pathogenic avian influenza (LPAI) viruses; however, the subtypes maintained in these populations and/or the transmission mechanisms involved are poorly understood. Although, a wide diversity of influenza viruses have been isolated from gulls, two hemagglutinin subtypes (H13 and H16) are rarely detected in other avian groups, and existing surveillance data suggests they are maintained almost exclusively within gull populations. In order to evaluate the host range of these gull-adapted influenza subtypes and to characterize viral infection in the gull host, we conducted a series of challenge experiments, with multiple North American strains of H13 LPAI virus in ring-billed gulls (Larus delawarensis), mallards (Anas platyrhynchos), chickens (Gallus domesticus), and turkeys (Meleagris gallopavo). The susceptibility to H13 LPAI viruses varied between species and viral strain. Gulls were highly susceptible to H13 LPAI virus infection and excreted virus via the oropharynx and cloaca for several days. The quantity and duration of shedding was similar between the two routes. Turkeys and ducks were resistant to infection with most strains of H13 LPAI virus, but low numbers of inoculated birds were infected after challenge with specific viral strains. Chickens were refractory to infection with all strains of H13 LPAI virus they were challenged with. The experimental results presented herein are consistent with existing surveillance data on H13 LPAI viruses in birds, and indicate that influenza viruses of the H13 subtype are strongly host-adapted to gulls, but rare spill-over into aberrant hosts (i.e., turkeys and ducks) can occur.

The use of hinged external fixation to provide additional stabilization for fractures of the distal humerus.

OBJECTIVE: To assess improvements in fixation stability when a hinged unilateral external fixator is used to supplement compromised internal fixation for distal humerus fractures. METHODS: Removing a 1-cm section of the distal humerus in cadaveric whole-arm specimens created a comminuted distal humerus fracture model (AO type 13-A3). Fixation was then performed using different constructs representing optimal, compromised, or supplemented internal fixation. Internal fixation consisted of either 2 reconstruction plates with 1, 2, or 3 (optimal) distal attachment screws, or crossing medial and lateral cortical screws. A hinged external fixator was applied in combination with compromised internal fixation. The stability of the different constructs was then evaluated using 3-point bending stiffness and distal fragment displacement measurements during flexion and extension testing. RESULTS: Addition of the external fixator increased the stiffness of all constructs. Stiffness of the compromised reconstruction plate constructs with supplemented fixation was similar to or significantly greater than that of optimal internal fixation. Addition of the fixator to the reconstruction plates with 1 screw or the crossing screws produced displacements of the distal fragment that were similar to those of the compromised constructs alone. However, medial/lateral and anterior/posterior displacements of the distal fragment during flexion and extension of the elbow for supplemented fixation were found to be greater than those for optimal internal fixation. CONCLUSIONS: The use of a hinged external fixator for supplemental fixation of distal humerus fractures may be effective in cases where internal fixation is severely compromised, although displacements may increase above optimal fixation.

Knee strength after total knee arthroplasty.

Fifty-two knees in normal healthy subjects and 32 knees more than 2 years after total knee arthroplasty (TKA) were evaluated. Average isometric extension peak torque values in TKA patients were reduced by up to 30.7% (P=.01). Isometric flexion peak torque values in patients with TKA were, on average, 32.2% lower than those from control subjects throughout the motion arc (P=.004). Knee Society Functional Scores were positively correlated to the average isometric extension peak torque (r=0.57; P=.004) and negatively correlated to the average isometric hamstring to quadriceps (H/Q) ratio (r=-0.78, P<.0001). Relatively greater quadriceps strength was associated with a better functional score. Older TKA patients (>/=70 years) generated lower isometric extension peak torque values in terminal extension than younger TKA patients (>24.2%; P=.05). Higher body mass index (BMI) was associated with relative quadriceps weakness (r=0.44; P=.007). These results suggest that more thorough rehabilitation after TKA would improve functional outcomes.

Average patient walking activity approaches 2 million cycles per year: pedometers under-record walking activity.

A two-dimensional accelerometer worn on the ankle (step activity monitor [SAM]; Prosthetic Research Study, Seattle, WA) has been proved to be highly accurate for assessing walking activity. The walking activity of 33 patients with well-functioning total hip arthroplasties was measured continuously during daily life, simultaneously with a pedometer and a SAM. The SAM recorded an average of 1.9 million cycles/y. The pedometer under-recorded an average of 34% cycles per day compared with the SAM (P=.0007), but the 2 measures were highly correlated (r=0.66; P=.001). No difference was seen in the number of gait cycles per day between men and women. The previously reported difference in average walking activity between men and women is due to greater under-recording of the pedometer in women, especially those with a body mass index > or =27. The pedometer is more reliable in quantifying the walking activity of men, less obese women, and patients with >1 million gait cycles/y.