Pathogens of free-ranging chickens create a risk of disease for wild birds, some of which migrate to the United States, as well as potential economic losses for resource-poor farmers. Free-roaming backyard chickens are commonly kept in shade-grown coffee plantations, habitats that attract large numbers of wild birds. The husbandry and pathogen prevalence of backyard chicken flocks in San Luis, Costa Rica, were investigated. Based on serologic evidence, Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus, chicken anemia virus, and infectious bursal disease virus, as well as both Mycoplasma gallisepticum and Mycoplasma synoviae, appear to be significant diseases of this population, and thus, we consider these backyard chickens potential reservoirs for these diseases. There was no evidence of avian influenza. Interviews, clinical examinations, and microscopic examination of tissues led us to believe that poxvirus is also a significant cause of morbidity and mortality in these chickens. We found that Escherichia coli isolates were resistant against tilmicosin, tetracycline, ampicillin, amoxicillin with clavulanic acid, ticarcillin, and cephalothin, and contained genes considered responsible for conferring tetracycline resistance. Additionally, although production was not measured, we suspect that husbandry and lack of preventative medicine are directly related to the diseases reported, all of which negatively affect production.

Abbreviations: AE = avian encephalomyelitis virus; AI = avian influenza; AMRP = antimicrobial resistance profile; APV = avian pneumovirus; CAV = chicken anemia virus; ELISA = enzyme-linked immunosorbent assay; IBD = infectious bursal disease; IBV = avian infectious bronchitis; ILT = infectious laryngotracheitis; MAG = Costa Rican Ministry of Agriculture; MIC = minimum inhibitory concentrations; MG = Mycoplasma gallisepticum; MS = Mycoplasma synoviae; NDV = Newcastle disease virus; PDRC = Population Diagnostic Research Center; PPMV = pigeon paramyxovirus; RT-PCR = real-time polymerase chain reaction; S/N ratio = signal-to-noise ratio; USDA = U.S. Department of Agriculture

Las parvadas de aves de traspatio representan un riesgo de enfermedad para las aves tropicales en Costa Rica.

Los patógenos de las aves de traspatio generan un riesgo de enfermedad para las aves silvestres, algunas de las cuales migran a los Estados Unidos, generando a su vez potenciales pérdidas económicas para granjeros de escasos recursos. Las aves de traspatio criadas a la intemperie son comúnmente mantenidas en plantaciones de café con abundante sombra, un habitat que atrae un gran número de aves silvestres. En San Luis, Costa Rica, se investigó el manejo y la prevalencia de patógenos en aves de traspatio. Basado en evidencia serológica, los virus de la enfermedad de Newcastle, laringotraqueitis infecciosa, bronquitis infecciosa, anemia infecciosa aviar y enfermedad infecciosa de la bolsa, así como el Mycoplasma gallisepticum y el Mycoplasma synoviae, son agentes causantes de enfermedades en esta población y en consecuencia se consideran a estas aves de traspatio como reservorios potenciales de estas enfermedades. No se encontró evidencia de influenza aviar. Entrevistas, exámenes clínicos y evaluaciones microscópicas de tejidos nos llevan a creer que el virus de la viruela aviar es también una causa significativa de morbilidad y mortalidad en estas aves. Se demostró que los aislamientos de Escherichia coli eran resistentes a la tilmicosina, tetraciclina, ampicilina, amoxiciclina y ácido clavulánico, ticarciclina, cefalocina y contenían genes consi

Young poultry are very susceptible to Salmonella Enteritidis (SE) infections because of the absence of complete intestinal flora colonization and an immature immune system. This study evaluated the role of passive immunity on the resistance of young birds against early infections caused by SE. The progeny of broiler breeders vaccinated with an oil-emulsion bacterin was compared to the progeny of unvaccinated birds. Efficacy was determined by challenging birds at 1 and 14 days of age with SE Nal Spc strain, phage type 4. After challenge at 1 day of age, the progeny of vaccinated birds presented a significantly lower number (log₁₀) of SE Nal Spc reisolation (P < 0.05) in liver (2.21), spleen (2.31), and cecal contents (2.85) compared with control groups (2.76, 3.02, and 6.03, respectively). The examination of the internal organs, 3 days after infection, revealed that 28% of the birds (7/25) from vaccinated breeders were positive, whereas 100% (25/25) of the chicks derived from unvaccinated birds were positive. Birds challenged at 14 days of age presented a lower number of positive samples compared with those challenged at 1 day of age, and the progeny of vaccinated birds presented statistically lower numbers (log₁₀) of colony-forming units/ml of SE Nal Spc only in the cecal contents compared with nonvaccinated breeder progeny (2.11 vs. 2.94). Age seems to influence the susceptibility of birds to SE infections: in control groups, the number of positive birds at 14 days of age (9/25) was lower when compared with the group infected at 1 day of age (25/25). The number of positive fecal samples of the progeny of vaccinated birds was significantly lower (36) than those of the control group (108) after challenge at 1 day of age. Unchallenged progeny of vaccinated birds presented passive antibodies detectable by enzyme-linked immunosorbent assay (ELISA) up to 21 days of age. On the other hand, antibodies of the control group were detected by ELISA 14 days after challenge. These results show a significant contribution of breeder vaccination by increasing the resistance of the progeny against early SE infections. However, the bacteria were not completely eliminated, suggesting that additional procedures are needed to effectively control SE infections.

Abbreviations: CFU = colony-forming units; dpi = days postinfection; ELISA = enzyme linked immunosorbent assay; Ig = immunoglobulin; Nal = nalidixic acid; SE = Salmonella Enteritidis; Spc = spectinomycin

Inmunidad pasiva de la progenie de reproductoras pesadas vacunadas con una bacterina emulsionada en aceite contra Salmonella Enteritidis.

Las aves jóvenes son muy susceptibles a las infecciones con Salmonella Enteritidis debido a la ausencia de una colonización completa por parte de la flora intestinal y a la inmadurez de su sistema immune. Este estudio evaluó el papel de la inmunidad pasiva en la resistencia de aves jóvenes contra las infecciones tempranas causadas por Salmonella Enteritidis. La progenie de reproductoras pesadas vacunadas con una bacterina emulsionada en aceite fue comparada con la progenie de aves no vacunadas. La eficacia fue determinada por el desafío con la cepa de Salmonella Enteritidis Nal Spec, fagotipo 4. Después del desafío al día de edad, la progenie de aves vacunadas presentó un número significantemente (P < 0.05) menor (log₁₀) de reaislamientos de Salmonella Enteritidis Nal Spec en hígado (2.21), bazo (2.31) y contenido cecal (2.85) cuando se comparó con los resultados obtenidos para el grupo control (2.76, 3.02 y 6.03, respectivamente). La evaluación de los órganos internos 3 días des

Marek's disease virus (MDV) infection in the eye was studied chronologically after inoculating 1-day-old chickens with a very virulent MDV strain, Md5. The ocular lesions could be classified as early lesions (6–11 days postinoculation [dpi]) and late lesions (26 and 56 dpi), based upon the location and severity of the lesions. The early lesions involved iris, ciliary body, and choroid layer, and were characterized by endothelial cell hypertrophy, vasculitis, and infiltration of lymphocytes (mainly CD8 ), plasma cells, macrophages, and heterophils. Expression of early MDV-antigen pp38 in the cells infiltrating choroid layer was detected as early as 11 dpi. Late lesions consisted of severe lymphohistiocytic uveitis, keratitis, pectenitis, vitreitis, retinitis, and segmental to diffuse retinal necrosis. Cell infiltration included macrophages, granulocytes, plasma cells, and both CD4 and CD8 cells of various sizes. Expression of early MDV-antigen pp38 was readily found within the retina, uveal tract, and corneal epithelium. No expression of late-antigen gB or oncoprotein meq was detected in any of the eyes examined. A second experiment was conducted to study the effect of vaccination on the development of ocular lesions. Both HVT and CVI988 were able to protect against the development of early ocular lesions in chickens infected with very virulent plus strain MDV 648A. However, only CVI988 conferred complete protection against the development of late ocular lesions. HVT conferred partial protection, as it reduced the frequency and severity of the late ocular lesions. These results enhance our understanding of the nature and pattern of MDV infection in the eye.

Abbreviations: ABC = avidin-biotin-peroxidase complex; CEF = chicken embryo fibroblasts; DEF = duck embryo fibroblasts; dpi = days postinoculation; H&E = hematoxylin and eosin; HSV = herpes simplex virus; HVT = herpesvirus of turkeys; MAb = monoclonal antibodies; MD = Marek's disease; MDV = Marek's disease virus; MHC = major histocompatibility complex; OCT = optimal cutting temperature; PN = peripheral neuropathy; S/C = subcutaneous route; SPAFAS = specific pathogen free; VZV = varicella-zoster virus

Infección en el ojo con el virus de la enfermedad de Marek: Estudio cronológico de lesiones, replicación viral y protección inducida mediante la vacunación.

La infección por el virus de la enfermedad de Marek se estudió cronológicamente luego de la inoculación de pollitos de un día de edad con una cepa muy virulenta del virus de la enfermedad de Marek denominada Md5. Basándose en la localización y severidad de las lesiones oculares estas pudieron ser clasificadas como lesiones tempranas (de seis a 11 días posteriores a la inoculación) y lesiones tardías (de 26 a 56 días posteriores a la inoculación). Las lesiones tempranas infectaron el iris, el cuerpo ciliar y la capa coroidea y se caracterizaron por hipertrofia de las células endoteliales, vasculitis e infiltración linfocitaria (principalmente linfocitos T CD8 ), células plasmáticas, macrófagos y heterófilos. La expresión del antígeno temprano pp38 del virus de la enfermedad de Marek en las células infiltrando la capa coroidea se detectó desde el día 11 posterior a la inoculación,. Las lesiones tardías consistían en uveítis linfohistiocítica severa, queratitis, pectenitis, vitreítis, retinitis y necrosis retinal que varió de segmentada a difusa. La infiltración celular incluía macrófagos, granulocitos, células plasmáticas y células CD4 y CD8 de varios tamaños. La expresión del antígeno temprano pp38 del virus de la enfermedad de Marek fue muy evidente en la retina, tracto uveal y epitelio de la córnea. En ninguno de los ojos examinados se detectó la expresión del antígeno tardío gB o de la oncoproteína meq. Se realizó un segundo experimento para estudiar el efecto de la vacunación en el desarrollo de las lesiones oculares. Tanto el virus Her

Listeria monocytogenes contaminates poultry processing plants due to its ubiquitous nature and high resistance to disinfection. The sources of the persistent, biofilm-forming strains that colonize processing plants are unknown. The purpose of this study is to determine if intrinsic colonization of turkeys is a possible source of contamination. Male poults were subjected to cold stress from 4–12 days of age; poults were unchallenged or were exposed to an aerosol and oral challenge of either an avian pathogenic strain of Escherichia coli (Ec), the Scott A strain of L. monocytogenes (Lm), or to a combination (Ec–Lm). At 7 wk, all cold-stressed poults were treated with an immunosuppressive dose of dexamethasone (Dex) and exposed to the same bacterial challenges. Birds were necropsied at 1 wk and 2 wk post-Dex treatment. Percent mortality, body and organ weights, and airsacculitis scores were determined. Liver and knee synovial tissues were sampled, using transport swabs, and cultured by direct plating, pre-enrichment, and Taqman® real-time PCR. There were no significant differences in cumulative mortality, and airsacculitis scores were variable but tended to be decreased by cold stress. Relative weights of liver and heart were increased, whereas body weights, and spleen and bursa relative weights, were decreased following all challenges. Listeria monocytogenes was isolated from up to 50% of liver or knee synovial tissues, using pre-enrichment followed by culturing methods, and from up to 67% of knee synovial tissues using pre-enrichment followed by real-time PCR at 1 wk postchallenge. The higher percentages of positive birds, detected by real-time PCR rather than by culture, and the negative results at 2 wk post-challenge suggest that the birds experienced a transient infection that was cleared by the immune system. These results suggest that environmentally acquired L. monocytogenes can transiently colonize the liver and synovial tissues of stressed turkeys and may be a sporadic source of contamination of processing plants.

Abbreviations: CBA = Columbia blood agar; CFU = colony forming unit(s); Dex = dexamethasone; Ec = Escherichia coli challenge; Ec–Lm = Listeria monocytogenes and Escherichia coli co-challenge; FAC = ferric ammonium citrate; LA = Listeria selective agar; Lm = Listeria monocytogenes challenge; R-ti-PCR = real-time polymerase chain reaction; TOC = turkey osteomyelitis complex; UVM = University of Vermont modified Listeria medium; UVM–I = University of Vermont enrichment media

Efecto del estrés en la enfermedad respiratoria y colonización transitoria con Listeria monocytogenes Scott A en pavos.

La Listeria monocytogenes contamina las plantas de procesamiento avícola debido a su naturaleza ubicua y alta resistencia a la desinfección,. Se desconocen las fuentes de las cepas persistentes formadoras de biocapas que colonizan las plantas de procesamiento. El propósito de este estudio fue determinar si la colonización intrínseca de los pavos representa una fuente posible de contaminación. Pavos machos jóvenes se expusieron a estrés por frío desde los cuatro hasta los 12 días de edad, las aves se mantuvieron sin desafío o fueron expuestas a un desafío oral y por aerosol con una cepa aviar de Escherichia coli patógena, la cepa Scott A de L. monocytogenes o a una combinación de ambas. A las siete semanas, todas las aves estresa

Infectious bursal disease virus (IBDV) serotype 1 is the causative agent of a highly contagious immunosuppressive disease of young chickens. In the past, a number of antigenic, as well as pathogenic, subtypes have been described. The determination of the antigenic makeup of circulating strains is of vital interest to the poultry industry because changes in the antigenicity of circulating field strains have an impact on the use of vaccines. To obtain a more comprehensive overview of the relationship between the nucleotide and amino acid sequence and the antigenic makeup of field isolates, a system based on reverse genetics of IBDV was established. Using this approach, a database for field isolates from three different states in the United States (Georgia, Alabama, and Louisiana), consisting of nucleotide sequence, amino acid sequence, and a reaction pattern based on a panel of monoclonal antibodies, was established. The obtained results showed that phylogenic analysis, which is based on the similarity of sequences, would lead to false conclusions regarding a possible antigenic makeup of the particular isolate. Sequences of field samples were divided into three groups: 1) those that grouped with variant strain E/Del sequences but were antigenically different, 2) those that did not group with sequences of E/Del but were similar in their antigenic makeup, and 3) those that did not group with E/Del sequences and were antigenically different. In addition, using the reverse-genetics approach, a number of field isolates showed no reactivity with any of the used monoclonal antibodies, indicating that an unknown, antigenic subtype of IBDV serotype I is circulating in the field.

Abbreviations: AC-ELISA = antigen-capture enzyme-linked immunosorbent assay; FITC = fluorescein isothiocyanate; IBDV = infectious bursal disease virus; MAb = monoclonal antibodies; NCR = noncoding region; nt = nucleotide; PBS = phosphate-buffered saline; PCR = polymerase chain reaction; QM = quail muscle; RE = restriction endonuclease; RFLP = restriction fragment length polymorphism; RT = reverse transcription; VN = virus neutralization

Detección por medio de genética reversa de aislados de virus de la enfermedad infecciosa de la bolsa con propiedades antigénicas desconocidas.

El serotipo 1 del virus de la enfermedad infecciosa de la bolsa de Fabricio es el agente causal de una enfermedad inmunosupreosora, altamente contagiosa en aves jóvenes. En el pasado se han descrito varios subtipos antigénicos y patogénicos de este virus. La determinación de la composición de cepas circulantes es de vital interés para la industria avícola porque los cambios en la antigenicidad de cepas de campo circulantes tienen un gran impacto en el uso de vacunas. Para obtener un panorama más comprensible de la relación entre las secuencias de nucleótidos y aminoácidos y la composición antigénica de los virus aislados de campo, se estableció un sistema basado en la genética reversa del virus de la enfermedad infecciosa de la bolsa. Usando este enfoque, se estableció una base de datos de los aislados de campo procedentes de tres estados diferentes de los Estados Unidos (Georgia, Alabama y Luisiana), consistente en la secuencia de nucleótidos, secuencia de aminoácidos y el patrón de reacción frente a un panel de anticuerpos monoclonales. Los resultados obtenidos mostraron que el análisis filogenético, basado en la similitud de las secuencias, pueden conducir a conclusiones falsas relacionadas con la composición antigénica de una cepa en particular. Las secuencias de los aislados de campo fueron divididas en tres grupos: 1) Aquellas agrupadas con la secuencia de la cepa variante E/Delaware pero que eran antigénicamente diferentes, 2) Aquellas que no se agruparon en la secuencia de la cepa E/Delaware pero que eran similares en su composición antigénica y 3) Aquellas que no se agruparon con las secuencias de la cep

An increasing prevalence of antibiotic-resistant foodborne infections has resulted in considerable concern about how antimicrobials are used in meat and poultry production. Because many foodborne bacterial pathogens are commonly found among the intestinal bacterial community of poultry, new methods of prevention are being considered. Bacteriophage therapy is one such alternative method that has not been well developed in the United States; however, bacteriophages have been shown to be effective in modulating bacterial numbers in acute infection models. In this study we evaluated whether bacteriophages could theoretically reduce Salmonella colonization of the gastrointestinal tract of chickens. Using computer simulations, we studied bacteriophage and bacterial replication dynamics in a mathematical model based on parameters expected to occur in the intestinal environment. In addition, we performed in vivo experiments by administering SP6 bacteriophage and Salmonella orally to young chickens and compared the levels of phage and Salmonella shed in the feces to the models of replication dynamics. SP6 is an ideal candidate bacteriophage because its genome and target receptor are known. Although SP6 did not reduce the levels of Salmonella shed by treated birds, most of the isolates recovered from treated birds were not resistant to the bacteriophage. These results suggest that phage resistance may not be the primary limiting parameter of phage prophylaxis for modulating colonization of the intestine. Our findings that this phage could be replicated in vivo supports the attractiveness of phage use, because unlike antibiotics they may be amplified in vivo if given a suitable host on which to replicate. If successful, this approach to modulating bacterial colonization of the intestinal tract could have a tremendous effect on the meat and poultry industry by reducing the use of antimicrobial drugs and increasing the use of biological therapeutics.

Abbreviations: BHI = brain heart infusion; bp = base pair; cfu = colony-forming units; LPS = lipopolysaccharide; PCR = polymerase chain reaction; pfu = plaque-forming units

Uso de bacteriófagos para modular la colonización de Salmonella en el tracto gastrointestinal de los pollos: Lecciones aprendidas con los modelos In Silico e In Vivo

El incremento en la prevalencia de la resistencia a los antibióticos en infecciones originadas en los alimentos ha resultado en una considerable preocupación acerca de la forma como los antimicrobianos son usados en la producción de carne. Debido a que muchos patógenos bacterianos son comúnmente encontrados entre la comunidad bacteriana de las aves, se están considerando nuevos métodos de prevención. La terapia con bacteriófagos es uno de los métodos alternos que no se han desarrollado bien en los Estados Unidos; sin embargo, los bacteriófagos han mostrado su efectividad en la modulación del número de bacterias en modelos de infecciones agudas. En este estudio evaluamos si los bacteriófagos pueden teóricamente reducir la colonización con Salmonella del tracto gastrointestinal de los pollos. Usando simulaciones computarizadas, estudiamos los bacteriófagos y la dinámica de replicación de las bacterias en un modelo matemático basado en parámetros que se espera ocurran en el ambiente intestinal. Adicionalmente, desarrollamos experimentos in vivo administrando bacteriófago SP6 y Salmonella oralmente a pollitos jóvenes y se compararon los niveles de fagos y Salmonella eliminados en las heces con los modelos de las dinámicas de replicación. El bacteriófago SP6 es un candidato ideal porque su genoma y su receptor objetivo son conocidos. Aunque

We compared detection of infectious bronchitis virus (IBV) by quantitative RT-PCR (qRT-PCR) in tears and trachea of IBV-infected chickens and found that quantitative detection of IBV RNA in tears is more sensitive than in tracheal homogenates. Furthermore, we demonstrated that IBV contained in chicken lachrymal fluid is infectious and that tears of IBV-infected chickens can be used to infect naive chickens. We compared the immune responses to IBV in the Harderian gland and cecal tonsils of immunocompetent chickens and chickens infected with chicken anemia virus (CAV) and/or infectious bursal disease virus (IBDV). Flow cytometry analyses of lymphocytes in Harderian glands and cecal tonsils indicated that the relative abundance of IgM B cells in the Harderian glands and cecal tonsils following exposure to IBV in combination with immunosuppressive viruses was reduced compared to chickens infected with IBV alone. CAV, but not IBDV, reduced the CD4 /CD8 T cell ratios compared to chickens infected with IBV alone. Enzyme-linked immuno-spot forming assays on cells in the Harderian glands and cecal tonsils of IBV-infected chickens indicated that maximum IBV-specific IgA-secreting cell responses were reduced in chickens infected with CAV. IBDV co-infected chickens displayed a delayed IgA response to IBV. Thus immunosuppressive viruses reduced B cells and T helper cells in the Harderian glands and cecal tonsils in response to IBV, and slowed the kinetics and/or reduced the magnitude of the mucosal immune response against IBV. We have shown for the first time that CAV affects pathogen-specific B cell responses in a mucosal effector site.

Abbreviations: APHIS = Animal Plant & Health Inspection Service; CAV = chicken anemia virus; CT = cecal tonsil; CTL = cytotoxic T-lymphocyte; DPI = days post-IBV inoculation; EID₅₀ = 50% embryo infective dose; ELISPOT = enzyme-linked immuno-spot forming; FACS = fluorescence-activated cell sorter; HG = Harderian gland; IBDV = infectious bursal disease virus; IBV = infectious bronchitis virus; Ig = immunoglobulin; PBS = phosphate buffer solution; PCR = polymerase chain reaction; qRT-PCR = quantitative RT-PCR; RT-PCR = reverse transcriptase PCR; SFC = spot-forming cells; SPF = specific-pathogen-free

Virus de bronquitis infecciosa en el fluido lacrimal y en la glándula de Harder del pollo: Carga viral, infectividad, respuestas inmune celular y efectos del virus.

Por medio de la prueba cuantitativa de la reacción en cadena por la polimerasa-transcripción reversa en tiempo real, se comparó la detección del virus de bronquitis infecciosa en las lágrimas y la tráquea de pollos infectados con el virus de bronquitis infecciosa y se encontró que la detección cuantitativa del ácido ribonucléico del virus en las lágrimas es más sensible que en los homogenados de la tráquea. Así mismo, se demostró que el virus de bronquitis presente en el fluido lagrimal es infeccioso y que las lágrimas de pollos infectados con el virus de bronquitis pueden usarse para infectar pollos susceptibles. Se comparó la respuesta inmune al virus de bronquitis infecciosa en la glándula de Harder y en las tonsilas cecales de pollos inmunocompetentes y en pollos infectados con el virus de anemia infecciosa aviar y con el de la enfermedad infecciosa de la bolsa (Gumboro), o con ambos. El análisis por medio de la citometría de flujo de los linfocitos en la glándula de Harder y en las tonsilas cecales indicó una reducción de la abundancia relativa de las células B IgM en estos dos órganos después de la exposición al virus de bronquitis en combinación con los virus inmunosupresores, comparado con los pollos infectados sólo con el virus de bronquitis. El virus de la anemia infecciosa, pero no el de Gumboro, redujo la proporción de las células T CD4 /CD8 , comparado con los pollos infectados

Among field isolates of infectious bronchitis virus (IBV) from recent outbreaks, some isolates that were classified into the 4/91 genotype, by analysis of the S1 gene, have been confirmed to be a new variant in Japan. To elucidate the characteristics of these isolates, pathogenicity in chicks and the efficacy of vaccines against this new variant were examined. Severe respiratory symptoms were observed in 4-day-old specific pathogen free chicks inoculated with a 4/91 genotype isolate, either JP/Wakayama/2003 or JP/Iwate/2005; body weights 3 wk after inoculation were significantly lower than those of chicks inoculated with a 4/91 vaccine strain. These 4/91 isolates were neutralized with serum from birds immunized with 4/91 vaccine. In a challenge–protection test, five groups of chicks were immunized with C78, TM-86w, H120, Kita-1, or 4/91 vaccines and then challenged with JP/Iwate/2005 4 wk after vaccination. A protective effect in the 4/91 and TM-86w vaccine groups was indicated by evaluation of the ciliostasis score of the trachea, the respiratory symptom score, and virus isolation from trachea swab samples after challenge. The results of this study suggested that the 4/91 type of IBV, which is virulent to chicks when compared to vaccine strains, has emerged as a new variant in Japan, and vaccines containing the 4/91 strain or the TM-86w strain could be effective for this variant.

Abbreviations: DPC = days postchallenge; DPI = days postinfection; EID₅₀ = 50% embryo infectious dose; HVR = hypervariable region; IB = infectious bronchitis; IBV = infectious bronchitis virus; PBS = phosphate buffered saline; RT-PCR = reverse transcriptase–polymerase chain reaction; SPF = specific pathogen-free; VN = virus-neutralizing

Aislamiento de un virus del tipo 4/91 como una nueva variante del virus de bronquitis infecciosa en Japón y eficacia de la vacunación contra una cepa de campo.

Entre los aislados de campo de virus de bronquitis infecciosa ocurridos recientemente, algunos aislados fueron clasificados como del genotipo 4/91, mediante el análisis del gen S1. Este tipo ha sido confirmado como una nueva variante en Japón. Para conocer las características de estas cepas, se evaluó la patogenicidad de las cepas en pollos y la eficacia de las vacunas. Cuando se inocularon aves libres de patógenos específicos de 4 días de edad con aislados genotipo 4/91, tanto JP/Wakayama/2003 como JP/Iwate/2005; se observaron severos síntomas respiratorios, el peso corporal 3 semanas postinoculación fue significativamente más bajo que el de aquellos pollos inoculados con una cepa vacunal 4/91. Estos aislados 4/91 fueron neutralizados con sueros provenientes de aves inmunizadas con una vacuna 4/91. En una prueba de protección al desafío, cinco grupos de pollos fueron inmunizados con vacunas C78, TM-86w, H120, Kita-1 ó 4/91 y desafiados 4 semanas después con la cepa JP/Iwate/2005. El efecto protector de las vacunas 4/91 y TM-86w fue evaluado mediante la calificación de la ciliostasis en la tráquea y los síntomas respiratorios, lo mismo que el aislamiento del virus a partir de muestras de hisopos traqueales tomadas después del desafío. Los resultados de este estudio sugieren que el virus de bronquitis infecciosa tipo 4/91, que es virulento para pollos cuando se compara con cepas vacunales, ha emergido como una nueva variante en el Japón, y las vacunas que contienen las cepas 4/91 ó la TM-86w pueden ser efectivas contra esta variante.

The virulence of low pathogenicity (LP) type A H7N2 avian influenza virus (AIV) isolates recovered from chickens in Delaware and the eastern shore of Maryland in 2004 was evaluated. Three-week-old leghorn- and broiler-type chickens and turkeys were inoculated via the conjunctival sac with 10^3.5–10^4.0 50% embryo infectious dose (EID₅₀) of virus per bird with A/chicken/Delaware/Viva/04, A/chicken/Delaware/Hobo/04, and A/chicken/Maryland/Minh Ma/04. In broilers, the viruses produced respiratory signs, airsacculitis, and microscopic lesions in the trachea and lung. In contrast, signs and lesions were less severe in turkeys, and they were rarely observed in specific-pathogen-free (SPF) leghorns. In broilers and SPF leghorns, AIV peaked on day 3 postinoculation (PI), based on virus isolation and real-time reverse transcription-polymerase chain reaction, and antigen capture testing. Infection in turkeys peaked on day 7 PI. Serum antibodies generally were detected earlier in broilers (day 7 PI) than in turkeys or SPF leghorns (day 14 PI) using agar gel immunodiffusion, hemagglutination-inhibition, and the enzyme-linked immunosorbent assay. A second trial was performed to further examine the disease susceptibility of the leghorn chicken given the comparatively mild responses noted in the first trial. A 10-fold higher dose of 10^4.5–10^5.0EID₅₀ per chick given via the conjunctival sac was used. In addition, commercial-type leghorns were tested as were chicks from the SPF leghorn source. The higher AIV dose resulted in more rapid and consistent rates of infection and higher serum antibody responses in both types of leghorn chickens. However, as observed in the first trial, clinical signs and microscopic lesions in both types of leghorns were infrequent and very mild. These findings indicate leghorn-type chickens, which are commonly used for pathogenicity assessments because of their availability, may not be the most suitable host for evaluating the virulence potential of LP AIV.

Abbreviations: AGID = agar gel immunodiffusion; AI = avian influenza; AIV = avian influenza virus; CAS = chorioallantoic sac; Ct = cycle threshold; EID₅₀ = embryo infectious dose₅₀; ELD₅₀ = embryo lethal dose₅₀; ELISA = enzyme-linked immunosorbent assay; HA = hemagglutination; HI = hemagglutination-inhibition; HP = high pathogenicity; LP = low pathogenicity; NVSL = National Veterinary Service Laboratory; PBS = phosphate-buffered saline; PI = postinoculation; RRT-PCR = real-time reverse transcription-polymerase chain reaction; SPF = specific-pathogen-free

Virulencia en pollos de engorde, aves leghorn y pavos, de aislados de virus H7N2 de influenza aviar de baja patogenicidad provenientes de la península de Delmarva.

Se evaluó la virulencia de aislamientos del virus de influenza aviar tipo A H7N2 aislados en el año 2004 a partir de aves domésticas en los estados de Delaware y la costa este de Maryland. Aves de la línea de postura (leghorn), pollos de engorde y pavos de tres semanas de edad fueron inoculados por vía ocular con una dosis infecciosa 50% para embrión de pollo de 10^3.5–10^4.0 de los siguientes virus: A/Pollo/Delaware/Viva/04, A/Pollo/Delaware/Hobo/04 y A/Pollo/Maryland/Minh Ma/04. En los pollos de engorde los virus produjeron signos respiratorios, aerosaculitis y lesiones microscópicas en la tráquea y pulmón. En contraste, las lesiones fueron menos severas en los pavos y fueron escasamente observadas en las aves leghorn libres de patógenos específicos. Mediante aislamiento viral, la prueba de reacción en cadena por la polimerasa transcriptasa reversa en tiempo real y pruebas de captura de antígenos, se determinó que en los pollos de engorde y en las aves libres de patógenos específicos los virus de influenza aviar alcanzaron un título máximo al terc

Two battery efficacy studies were conducted with field isolates of Eimeria spp. from outbreaks of coccidiosis on pheasant-rearing farms in Georgia (GA) and Illinois (IL), United States. The coccidian isolates were mixed species containing E. phasiani, E. duodenalis, and E. colchici. Lasalocid (LAS) was tested prophylactically at 120 parts per million (ppm) in the feed. Anticoccidial activity was assessed, in comparisons between infected and uninfected control birds, by the effects on weight gain and oocyst passage. The GA isolate reduced weight gain of infected controls by 64% (P < 0.05) during the week after inoculation. LAS-treated birds also suffered some depressed gain, but average gains were significantly improved (P < 0.05) in comparison with that of the infected controls. Fecal oocyst counts of infected controls averaged 10–24 × 10⁶/day for days 5–11 postinoculation (PI), after which oocyst passage declined. Birds given LAS passed 10–20 × 10⁶ oocysts on days 5–6 PI, after which counts dropped to much lower levels. Oocyst passage was significantly reduced (P < 0.05) by LAS treatment on all but days 5 and 6 PI. The IL isolate reduced weight gain by 36% in infected controls, relative to uninfected controls (P < 0.05). LAS-treated birds also suffered reduced gain, but the gain was significantly improved in comparison with the infected controls. Oocyst counts averaged 3–9 × 10⁶/day on days 6–14 PI, but were significantly reduced by LAS treatment on all but day 6 PI. Thus, LAS was effective in pheasants against the tested field isolates of Eimeria spp. and was well tolerated at 120 ppm in the feed. However, the level of control was not as high as previously observed. Possible explanations for this difference include an emergence of drug resistance from prior use of LAS and a differential efficacy of LAS against different species of Eimeria used in the studies.

Abbreviations: GA = Georgia; IL = Illinois; LAS = lasalocid; PI = postinoculation; ppm = parts per million

Eficacia del Lasolacid contra la coccidiosis en faisanes Chinos de cuello anillado.

Se realizaron dos estudios de eficacia con aislamientos de Eimeria provenientes de brotes de coccidiosis en granjas de cría de faisanes en los estados de Georgia e Illinois en los Estados Unidos. Los aislamientos de coccidia representaron una mezcla de especies: E. phasiani, E. duodenalis y E. colchici. El Lasolacid se ensayó profilácticamente en el alimento a una dosis de 120 partes por millón (ppm). La actividad anticoccidial se evaluó mediante la comparación de los efectos de las coccidias en la ganancia de peso y en la tasa de eliminación de ooquistes en aves control infectadas y no infectadas. El aislamiento de Georgia redujo la ganancia de peso de los controles infectados en un 64% (P < 0.05) durante la primera semana post-inoculación. Las aves tratadas con Lasolacid también sufrieron alguna depresión en la ganancia de peso, sin embargo, el promedio de las ganancias se mejoró significativamente (P < 0.05) en comparación con la ganancia de los controles infectados. Los recuentos de ooquistes fecales de los controles infectados promediaron 10–24 × 10⁶ ooquistes por día los días 5 a 11 post-inoculación, después de este periodo la eliminación de ooquistes declinó. Las aves tratadas con Lasolacid eliminaron 10–20 × 10⁶ ooquistes por día entre los días cinco y seis post inoculación, luego de los cuales los recuentos disminuyeron a niveles mucho más bajos. La eliminación de ooquist

Ulcerative enteritis-like disease due to Clostridium perfringens type A was attributed as the cause of mortality in excess of 50% in a flock of 1000, 10-to-16-wk-old bobwhite quail (Colinus virginianus). Clinical signs in these birds ranged from sudden death to listlessness, depression, watery white droppings, ruffled feathers, loss of weight, and death in a few days. Necropsy of 30 birds revealed multiple deep ulcers of the mucosa throughout the small intestine and ceca, some with perforation and subsequent coelomitis (peritonitis). The livers in some birds contained white foci of necrosis, and many birds had enlarged and congested spleens. Microscopic lesions included multifocal severe fibrinosuppurative ulcerative enteritis associated with large numbers of rod-shaped gram-positive bacteria, and necrotizing hepatitis with or without rod-shaped bacteria. Anaerobic culturing of the intestine and liver yielded pure cultures of C. perfringens. The C. perfringens isolates were of genotype A and were polymerase chain reaction-positive for α toxin and for cpb2, the structural gene for β2 toxin. Repeated attempts to isolate C. colinum by using a specialized medium containing 8% horse plasma were not fruitful, suggesting that the enteritis and hepatitis in these birds were produced by C. perfringens. Retrospective examination of records of quail submissions to the California Animal Health and Food Safety Laboratory System over 16 yr revealed at least nine quail submissions in which isolation of C. perfringens from the liver, intestine, or both was associated with ulcerative enteritis and hepatitis in quail. This is the first description of ulcerative enteritis-like disease in quail associated with C. perfringens. Final conclusions await experimental reproduction of the disease.

Abbreviations: APMV = avian paramyxovirus; CAHFS = California Animal Health and Food Safety Laboratory System; CAMP = Christie, Atkins, Munch-Peterson; CPE = Clostridium perfringens enterotoxin; CPBE = Clostridium perfringens β2 toxin; FTG = fluid thioglycollate; H&E = hematoxylin and eosin; HPLC = high-performance liquid chromatography; PCR = polymerase chain reaction; PEA = phenyl ethyl alcohol; SDS-PAGE = sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Enfermedad parecida a la enteritis ulcerativa asociada con Clostridium perfringens tipo A en codornices Virginianas (Colinus virginianus)

La enfermedad parecida a la enteritis ulcerativa asociada con Clostridium perfringens tipo A se consideró la causa de una mortalidad superior al 50% en una parvada de 1000 codornices Virginianas de entre diez y 16 semanas de edad. Los signos clínicos en estas aves variaron en un rango desde muerte súbita hasta depresión, heces acuosas blanquecinas, plumas erizadas, pérdida de peso y muerte en pocos días. La necropsia de 30 aves reveló múltiples úlceras profundas de la mucosa a lo largo del intestino delgado y el ciego, algunas con perforaciones y consecuente celomitis (peritonitis). Los hígados de algunas de las aves contenían focos blancos de necrosis y muchas aves mostraron bazos agrandados y congestionados. Las lesiones microscópicas incluyeron enteritis ulcerativa fibrino-supurativa multifocal severa asociada con numerosos bacilos Gram positivos y hepatitis necrotizante con o sin la presencia de los bacilos. El cultivo anaerób

The lesser kestrel (Falco naumanni) is one of the most endangered bird species in Europe, and a captive breeding and reintroduction project was established. A breeding project is vulnerable to pathogens, e.g., mycoplasmas, reducing the reproductive success and carrying the risk to release pathogens with the birds to the wild. Therefore, 18 infertile eggs and 43 dead in shell embryos of the breeding project, as well as 27 nestlings and 34 adult birds of the captive and three different free-ranging populations were investigated for the occurrence of mycoplasmas by culture and a Mycoplasma genus–specific polymerase chain reaction. All eggs, embryos, and hand-reared nestlings from the captive group were negative. In contrast, all parent-reared nestlings and 88% of the adults were positive. Mycoplasma falconis and unidentifiable mycoplasmas were detected in all groups. Mycoplasma buteonis was found in the captive and only in two of the three free-ranging populations. Sequencing the 16S rRNA gene of six randomly selected unidentified isolates showed that five isolates were similar and most likely had been found previously in a falcon from Germany. The remaining isolate demonstrated a very high homology to unidentified Mycoplasma isolates obtained previously from semen samples of raptors. The results suggest that these isolates might represent two new species. Mycoplasmas seem not to play a major role as pathogens in the breeding project, and there is no evidence that releasing birds poses a risk to the free-ranging population with regard to mycoplasmas. The study seems to be the first to describe the occurrence and role of mycoplasmas in the lesser kestrel.

Abbreviations: IBA = immunobinding assay; PCR = polymerase chain reaction; 16S rRNA = 16S ribosomal ribonucleic acid; UAE = United Arab Emirates

El papel de los micoplasmas en el proyecto de conservación del cernícalo primilla (Falco naumanni).

El cernícalo primilla (Falco naumanni) es una de las especies de aves en mayor peligro de extinción en Europa, por lo que se estableció un proyecto de reproducción en cautiverio y reintroducción. Un proyecto de reproducción en cautiverio es vulnerable a patógenos como por ejemplo los micoplasmas, reduciendo el éxito reproductivo y llevando el riesgo de liberar patógenos a las áreas silvestres conjuntamente con las aves. En consecuencia, se investigaron 18 huevos infértiles y 43 embriones muertos del proyecto de reproducción, así como 27 polluelos y 34 aves adultas cautivas y provenientes de tres poblaciones de crianza en libertad, para detectar la ocurrencia de micoplasmas mediante el cultivo y la prueba de reacción en cadena por la polimerasa específica del género Mycoplasma. Todos los huevos, embriones y polluelos criados en forma separada provenientes del grupo cautivo resultaron negativos. En contraste, todos los polluelos criados por sus padres y el 88% de las aves adultas resultaron positivos. Se detectó el Mycoplasma falconis y micoplasmas no identificables en todos los grupos. El Mycoplasma buteonis se encontró en las aves cautivas y sólo en dos de las tres poblaciones libres. La secuenciación del ARN del gen 16S de seis aislamientos no identificados seleccionados aleatoriamente, mostró que cinco de los aislamientos eran similares entre sí y muy probablemente habían sido hallados previamente en un halcón proveniente de Alemania. El aislamiento restante mostró una alta homología con aislamientos de micoplasma no identificados obtenidos previamente de muestras de semen de aves de caza. Los resultados sugieren qu

Muscadine pomace (MP), a by-product of the production of wine and juice from Vitis rotundifolia, was dried and tested in chickens for effects on primary resistance to coccidiosis, development of protective immunity after vaccination with live coccidia, and resistance to necrotic enteritis (NE) caused by the joint action of Clostridium perfringens and coccidia. To test primary resistance to coccidiosis, 2-wk-old chicks were given 2% or 5% MP in the diet and inoculated with Eimeria acervulina and E. maxima. Birds given MP at either level had significantly (P < 0.05) lower lesion scores at 7 days postinoculation, in comparison with control birds, although weight gains were statistically similar. Broiler chickens were given 2% or 5% MP and grown to 42 days to test the palatability of MP. Birds given 2% MP in feed grew similarly to untreated controls, but birds given 5% had poorer average live weight. This suggested a negative effect on feed intake at the higher level. The effects of dietary 0.5% or 2.0% MP on immune protection were tested after live coccidiosis vaccination in the hatchery. Chicks were removed from each pen at 21 days of age and challenged with E. acervulina, E. maxima, and E. tenella. Resistance to infection was improved by MP as suggested by significantly (P < 0.05) lower lesion scores 7 days postchallenge, and improved weight gains in comparison with immunized control birds that did not receive MP. At 42 days of age, birds given MP had higher average live weights than controls, although feed efficiency was not affected. An established model was used to study the effect of MP on NE in broiler chickens. Chicks were inoculated with live coccidia at 14 days of age and dosed orally with live cultures of C. perfringens on day 19, day 20, and day 21. Enteritis caused 48% mortality in the first study and 67% mortality in the second study. Dietary MP at 0.5–2.0% significantly (P < 0.05) reduced mortality in both experiments; improved weight gain relative to the unmedicated, infected control; and reduced lesion scores at necropsy. Overall, the results of six experiments suggested that MP given in the diet at 0.5% or higher had a positive effect on primary resistance and development of acquired resistance to two severe intestinal diseases in chickens.

Abbreviations: BMD = bacitracin-methylene disalicylate; CFU = colony-forming unit; CP = Clostridium perfringens; FC = feed conversion; IL = interleukin; ME = metabolizable energy; MP = muscadine pomace; NCR = National Research Council; NE = necrotic enteritis; PI = postinoculation

Aumento de la resistencia a la coccidiosis y la enteritis necrótica en pollos de engorde usando pulpa de uvas Muscadinas en la dieta.

La pulpa de uvas muscadinas es un producto de la producción del vino y del jugo de Vitis rotundifolia, fue deshidratado y ensayado en pollos para evaluar sus efectos en la resistencia primaria contra la coccidiosis, el desarrollo de la inmunidad protectiva después de la vacunación con coccidia viva y la resistencia a la enteritis necrótica causada por la acción conjunta de Clostridium perfringens y coccidia. Para evaluar la resistencia primaria a la coccidiosis, a pollitos de dos semanas de edad se les suministró 2% ó 5% de la pulpa de uvas en la dieta y posteriormente fueron inoculados con Eimeria acervulina y E. maxima. Las aves a las que se les suministró el producto en ambos niveles mostraron una disminución

To detect five different avian Eimeria species, we applied the SYBR Green-based real-time polymerase chain reaction (PCR) assay for the diagnosis of field-isolated parasites by using their individual species-specific primer sets. The primer sets were originally designed for Eimeria acervulina, E. brunetti, E. necatrix, and E. tenella based on the sequence of the internal transcribed spacer 1 region of ribosomal DNA, whereas for E. maxima the primer sets were derived from sequences reported previously. The detection limit of these assays was defined at 10² or 10¹ oocysts depending on species. Melting curves from the real-time PCR assay showed that each species has a single peak and specific melting temperature value. Fecal samples from 32 poultry farms, which were endemic for coccidiosis, were examined using this assay. The data showed that E. brunetti was found in 21 farms, E. maxima and E. necatrix in 16 farms, E. tenella in 12 farms, and E. acervulina in eight farms. This survey revealed that E. brunetti was highly prevalent in Japan. This technique is not only easy and rapid but also available to detect Eimeria species specifically; therefore, it can be a valuable tool for diagnostic work for chicken coccidiosis.

Abbreviations: bp = base pair(s); EA = Eimeria acervulina; EB = Eimeria brunetti; EM = Eimeria maxima; EN = Eimeria necatrix; ET = Eimeria tenella; ITS = internal transcribed spacer; OPG = oocysts per gram; PCR = polymerase chain reaction; rRNA = ribosomal RNA; Tm = melting temperature

Detección de cinco especies de Eimerias aviares mediante la prueba de reacción en cadena por la polimerasa en tiempo real específica de especie.

Para detectar cinco especies diferentes de Eimeria, se utilizó el método SYBR Green, basado en la prueba de reacción en cadena por la polimerasa (PCR por sus siglas en Inglés) en tiempo real para el diagnóstico de parásitos aislados de casos de campo usando grupos de iniciadores individuales específicos de especie. Los iniciadores fueron originalmente diseñados para E. acervulina, E. brunetti, E. necatrix, y E. tenella basados en la transcripción de la secuencia del espacio de la región interna 1 del DNA ribosomal, mientras que para E. maxima los iniciadores se derivaron de secuencias reportadas anteriormente. La detección límite de esta prueba fue definida como 10² ó 10¹ ooquistes dependiendo de la especie. El punto de fusión de las curvas del PCR en tiempo real mostró que cada especie tiene un pico particular o único y un valor de temperatura del punto de fusión específico. Con esta prueba se examinaron muestras de heces de 32 granjas avícolas comerciales que eran endémicas para coccidiosis. Los resultados mostraron que E. brunetti fue encontrada en 21 granjas, E. maxima y E. necatrix en 16, E. tenella en 12 y E. acervulina en 8. Esta encuesta reveló que