M. França

Influenza Pathobiology and Pathogenesis in Avian Species

Wild birds in the orders Anseriformes and Charadriiformes are the natural and asymptomatic reservoirs of influenza A viruses representing all of the avian hemagglutinin (HA) and neuraminidase (NA) subtypes. Transmission of avian influenza (AI) viruses from wild birds to gallinaceous poultry species occurs regularly and outcomes vary, ranging from asymptomatic infections to mortality. Circulation of H5 and H7 low pathogenic AI (LPAI) viruses in gallinaceous poultry may result in mutations in the HA protein cleavage site and the emergence of highly pathogenic AI (HPAI) viruses, which in poultry can cause severe disease with high economic losses. Since 2002, various wild bird species also have succumbed to infection with the Eurasian H5N1 HPAI viruses. The pathogenesis of AI is complex and the ability of these viruses to produce disease and death in avian species is dependent on various host, viral and environmental factors, which are not completely understood.

Ulcerative Enteritis-like Disease Associated with Clostridium sordellii in Quail

SUMMARY A natural outbreak of ulcerative enteritis-like disease associated with Clostridium sordellii was diagnosed in two commercial quail flocks. Clinical signs in the quail included anorexia, weakness, and increased mortality in the flocks. Lesions in the intestine were characterized by ulcers covered with fibrinonecrotic exudate in the small intestine and occasional hemorrhages. There were also multifocal pale areas of necrosis in the liver. Clostridium sordellii was isolated from the intestine and liver. A retrospective study of avian cases submitted to the California Animal Health and Food Safety Laboratories revealed that C. sordellii had been isolated in 45 avian submissions, most commonly in chickens and turkeys. In most of these cases the birds were diagnosed with necrotic enteritis, with or without hepatitis. Clostridium sordellii has occasionally been associated with gangrenous dermatitis in poultry, but this is the first report of enteritis in an avian species. RESUMEN Reporte de Caso—Enfermedad similar a la enteritis ulcerativa asociada con Clostridium sordellii en codornices. Se diagnosticó un brote natural de una enfermedad similar a enteritis ulcerativa asociada con Clostridium sordellii en dos parvadas comerciales de codornices. Los signos clínicos en las codornices incluyeron anorexia, debilidad y mortalidad aumentada en las parvadas. Las lesiones en el intestino estuvieron caracterizadas por úlceras cubiertas con exudado fibrinonecrótico en el intestino delgado y hemorragias ocasionales. En el hígado se observaron áreas pálidas de necrosis. Se aisló Clostridium sordellii del intestino y del hígado. Un estudio retrospectivo de los casos aviares remitidos a los Laboratorios de Salud Animal e Inocuidad de los Alimentos de California, reveló que se había aislado C. sordellii en 45 casos aviares, más comúnmente en pollos y pavos. En la mayoría de los casos, las aves fueron diagnosticadas con enteritis necrótica, con o sin la presencia de hepatitis. Ocasionalmente, Clostridium sordellii ha sido asociado con dermatitis gangrenosa en las aves comerciales, pero este es el primer reporte de enteritis en una especie aviar.

The Pathogenesis of Low Pathogenic Avian Influenza in Mallards

SUMMARY. Mallards are important natural hosts involved in the epidemiology of low pathogenic avian influenza viruses (LPAIVs). LPAIVs are mainly transmitted by a fecal-oral route and are excreted in high concentrations in the feces. We investigated the pathology, viral antigen distribution, and the expression of &agr;2,3 sialic acid (SA) influenza virus receptors in mallards after intranasal inoculation with A/Mallard/MN/199106/99 (H3N8) or A/Mallard/MN/355779/00 (H5N2). Gross lesions were not observed. Avian influenza virus (AIV) nucleoprotein (NP) antigen was detected in rare epithelial cells of the larynx and trachea only at 1-day postinoculation (dpi) in the birds infected with H3N8 LPAIV, but infection with either virus was associated with lymphocytic tracheitis and laryngitis on 1 and 2 dpi. AIV NP antigen was detected in enterocytes of the lower intestine from 1 to 4 dpi and in epithelial cells of the bursa of Fabricius from 2 to 3 dpi in birds infected with either virus. Oropharyngeal and cloacal viral shedding was detected from 1 dpi, with higher cloacal viral shedding detected at 2 and 3 dpi with both viruses. Mallards abundantly expressed &agr;2,3 sialic acid receptors in epithelial cells of the respiratory tract, lower intestine, and bursa of Fabricius. Some infected birds had decreased &agr;2,3 sialic acid expression in epithelial cells of the bursa of Fabricius and in enterocytes of the ceca and colon. In conclusion, the main sites of LPAIV replication in mallards are the enterocytes of the lower intestinal tract and epithelial cells of the bursa of Fabricius in the first days after infection, when these birds are shedding AIV in high titers in the feces.

Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus

Waterfowl are considered the natural reservoir of low-virulence Newcastle disease viruses (loNDVs) and low-pathogenic avian influenza viruses (LPAIVs). The objective of this study was to investigate the effect of co-infections with loNDV and LPAIV on the infectivity and excretion of these viruses in mallards. One-month-old mallards were inoculated intranasally with 106 median embryo infectious doses of a wild-bird-origin loNDV and A/Mallard/MN/199106/99 (H3N8) LPAIV on the same day or received the LPAIV 2 or 5 days after loNDV inoculation. All mallards became infected with both viruses based on detection of seroconversion and viral shedding. Co-infection resulted in a higher number of cloacal swabs detected positive for LPAIV and a lower number of cloacal swabs detected positive for loNDV in some groups, although differences between groups were not statistically significant. Co-infection did not affect replication of LPAIV in epithelial cells of the lower intestine and bursa of Fabricius. In summary, the results of this study indicate that co-infection with LPAIV and loNDV does not affect the ability of mallards to be infected with either virus although it may have minimal effects on patterns (source and timing) of viral shedding.

Effect of Different Routes of Inoculation on Infectivity and Viral Shedding of LPAI Viruses in Mallards

SUMMARY. We studied the effect of different routes of inoculation on the infectivity and duration of viral shedding in mallards (Anas platyrhynchos) infected with wild bird-origin low pathogenic avian influenza viruses (LPAIVs). One-month-old mallards were inoculated with 106 median embryo infectious doses of either A/mallard/MN/199106/99 (H3N8) or A/mallard/MN/355779/00 (H5N2) via 1 of 5 different routes: intranasal (IN), intratracheal (IT), intraocular (IO), intracloacal (IC), or intra-ingluvial (II). Birds in all routes of inoculation groups became infected with LPAIV as detected by virus isolation, real time reverse transcription polymerase chain reaction, and serology. Mallards in different route of inoculation groups had similar viral shedding through oropharynx and cloaca from 1 day postinoculation (dpi). The peak of oropharyngeal (OP) viral shedding was reached between 2 and 3 dpi in all routes of inoculation groups infected with either virus. The peak of cloacal (CL) viral excretion was reached between 2 and 3 dpi in all routes of inoculation groups infected with H3N8 LPAIV and in the IO-, IC-, and II-inoculated groups infected with H5N2 LPAIV, with a delayed and shorter peak for the IN- and IT-inoculated groups. The birds inoculated via the II route had more productive OP and CL viral shedding after infection with either LPAIV, as evidenced by higher number of swabs testing positive over the study period. In conclusion, mallards can be infected with LPAIV by various routes of inoculation, and this corroborates their high susceptibility to infection by these viruses.

Adaptive Heterosubtypic Immunity to Low Pathogenic Avian Influenza Viruses in Experimentally Infected Mallards.

Mallards are widely recognized as reservoirs for Influenza A viruses (IAV); however, host factors that might prompt seasonality and trends in subtype diversity of IAV such as adaptive heterosubtypic immunity (HSI) are not well understood. To investigate this, we inoculated mallards with a prevailing H3N8 low pathogenic avian influenza virus (LPAIV) subtype in waterfowl to determine if prior infection with this virus would be protective against heterosubtypic infections with the H4N6, H10N7 and H14N5 LPAIV subtypes after one, two and three months, respectively. Also, we investigated the effect of cumulative immunity after sequential inoculation of mallards with these viruses in one-month intervals. Humoral immunity was assessed by microneutralization assays using a subset of representative LPAIV subtypes as antigens. Our results indicate that prior inoculation with the H3N8 virus confers partial protective immunity against subsequent heterosubtypic infections with the robustness of HSI related to the phylogenetic similarity of the HA protein of the strains used. Furthermore, induced HSI was boosted and followed by repeated exposure to more than one LPAIV subtype. Our findings provide further information on the contributions of HSI and its role in the dynamics of IAV subtype diversity in mallards.

Competition between influenza A virus subtypes through heterosubtypic immunity modulates re-infection and antibody dynamics in the mallard duck

Our overall hypothesis is that host population immunity directed at multiple antigens will influence the prevalence, diversity and evolution of influenza A virus (IAV) in avian populations where the vast subtype diversity is maintained. To investigate how initial infection influences the outcome of later infections with homologous or heterologous IAV subtypes and how viruses interact through host immune responses, we carried out experimental infections in mallard ducks (Anas platyrhynchos). Mallards were pre-challenged with an H3N8 low-pathogenic IAV and were divided into six groups. At five weeks post H3N8 inoculation, each group was challenged with a different IAV subtype (H4N5, H10N7, H6N2, H12N5) or the same H3N8. Two additional pre-challenged groups were inoculated with the homologous H3N8 virus at weeks 11 and 15 after pre-challenge to evaluate the duration of protection. The results showed that mallards were still resistant to re-infection after 15 weeks. There was a significant reduction in shedding for all pre-challenged groups compared to controls and the outcome of the heterologous challenges varied according to hemagglutinin (HA) phylogenetic relatedness between the viruses used. There was a boost in the H3 antibody titer after re-infection with H4N5, which is consistent with original antigenic sin or antigenic seniority and suggest a putative strategy of virus evasion. These results imply competition between related subtypes that could regulate IAV subtype population dynamics in nature. Collectively, we provide new insights into within-host IAV complex interactions as drivers of IAV antigenic diversity that could allow the circulation of multiple subtypes in wild ducks.

Polymorphisms in the S1 spike glycoprotein of Arkansas-type infectious bronchitis virus (IBV) show differential binding to host tissues and altered antigenicity

Abstract Sequencing avian infectious bronchitis virus spike genes re-isolated from vaccinated chicks revealed that many sequence changes are found on the S1 spike gene. In the ArkDPI strain, Y43H and ∆344 are the two most common changes observed. This study aims to examine the roles of Y43H and ∆344 in selection in vivo. Using recombinant ArkDPI S1 proteins, we conducted binding assays on chicken tracheas and embryonic chorioallantoic membrane (CAM). Protein histochemistry showed that the Y43H change allows for enhanced binding to trachea, whereas the ArkDPI S1 spike with H43 alone was able to bind CAM. Using Western blot under denaturing conditions, ArkDPI serotype-specific sera did not bind to S1 proteins with ∆344, suggesting that ∆344 alters antigenicity of S1. These findings are important because they propose that specific changes in S1 enhances virus fitness by more effective binding to host tissues (Y43H) and by evading a vaccine-induced antibody response (∆344).

Nephritis Associated with Infectious Bronchitis Virus Cal99 Variant in Game Chickens

SUMMARY. Infectious bronchitis virus (IBV) Cal99 variant was isolated from the kidneys of seven 2–5-mo-old game chickens with nephritis and respiratory disease. IBV Cal99 variant is usually associated with respiratory disease in broiler chickens in California. Macroscopically, the majority of the birds had moderately to severely enlarged and mottled pale kidneys, with increased urates in the ureters. Microscopically, most of the birds had acute nephrosis and interstitial nephritis. The birds also had sinusitis, tracheitis, bronchopneumonia, airsacculitis, salivary gland adenitis, and lymphoid depletion in the thymus and bursa of Fabricius. Immunohistochemistry was strongly positive for IBV antigen in the cytoplasm of tubular epithelial cells in the kidneys and also in the epithelium of the respiratory tract, salivary glands, proventriculus, intestine, and bursa of Fabricius. Infectious bronchitis virus was isolated from the trachea, lungs, kidneys, and cecal tonsils. Sequencing of the hypervariable region of the S1 gene of the kidney IBV isolate, designated IBV/CA99variant/07, revealed that the virus was 98% homologous to the Cal99 serotype of IBV.

Retrospective Study of Myocarditis Associated With Reovirus in Turkeys

Abstract The myocarditis associated with reovirus in commercial turkeys was studied retrospectively. Fifty-two cases were identified between 1991 and 2009. The lesions occurred in four different poultry companies in California and affected male and female turkeys with an average age of 19 days. Increased mortality in the turkey flocks ranged from 0.35% to 3% per week in 47 cases. Reovirus was isolated from the heart in 14 out of 19 cases. Twenty-four out of 28 birds from nine cases had low vitamin E levels in the liver ranging from 0.29 to 2.5 ppm (normal 3.0 to ≥15 ppm). Transmission electron microscopy of the heart revealed degenerative changes in the myocardial cells. Reovirus has been suggested as a probable etiology of this condition. Vitamin E deficiency might also contribute to the development of the lesions.