Observations about declining fertility in a feline breeding colony.

Feline breeding colonies are important to the feline industry by preserving traits desirable for a particular breed or in research settings by maintaining medically valuable genetic traits. As breeding females age, their reproductive efficiency declines. The objective of this study was to determine the most common causes of infertility in breeding females that were producing fewer kittens. Knowing the cause and average age of infertility would allow management decisions to be made for the betterment of the colony. The medical records of 70 queens retired from breeding from a single research colony were examined for litter size and number, fertility over their lifespan, and age and reason for removal from breeding stock. Sections of uterus and ovaries were evaluated using gross and histopathological examination for a subset of these queens (46). The data suggests that mature, continuously breeding female cats may show signs of reduced fertility (infertile matings or reduced litter size) as early as 3 years of age and may be a result of undiagnosed Cystic Endometrial Hyperplasia (CEH), endometritis, pyometra and/or ovarian cysts. Evaluation of breeding queens should include periodic ultrasounds to monitor for ovarian cysts and evidence of CEH. Retiring animals from breeding once signs of infertility become apparent is recommended.

Enhanced Protection by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Virus Spike Ectodomain and Chicken Granulocyte-Macrophage Colony-Stimulating Factor.

We previously reported that recombinant Newcastle disease virus LaSota (rLS) expressing infectious bronchitis virus (IBV) Arkansas (Ark)-type trimeric spike (S) ectodomain (Se; rLS/ArkSe) provides suboptimal protection against IBV challenge. We have now developed rLS expressing chicken granulocyte-macrophage colony-stimulating factor (GMCSF) and IBV Ark Se in an attempt to enhance vaccine effectiveness. In the current study, we first compared protection conferred by vaccination with rLS/ArkSe and rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with virulent Ark, and protection was determined by clinical signs, viral load, and tracheal histomorphometry. Results showed that coexpression of GMCSF and the Se from rLS significantly reduced tracheal viral load and tracheal lesions compared with chickens vaccinated with rLS/ArkSe. In a second experiment, we evaluated enhancement of cross-protection of a Massachusetts (Mass) attenuated vaccine by priming or boosting with rLS/ArkSe.GMCSF. Vaccinated chickens were challenged with Ark, and protection was evaluated. Results show that priming or boosting with the recombinant virus significantly increased cross-protection conferred by Mass against Ark virulent challenge. Greater reductions of viral loads in both trachea and lachrymal fluids were observed in chickens primed with rLS/ArkSe.GMCSF and boosted with Mass. Consistently, Ark Se antibody levels measured with recombinant Ark Se protein-coated ELISA plates 14 days after boost were significantly higher in these chickens. Unexpectedly, the inverse vaccination scheme, that is, priming with Mass and boosting with the recombinant vaccine, proved somewhat less effective. We concluded that a prime and boost strategy by using rLS/ArkSe.GMCSF and the worldwide ubiquitous Mass attenuated vaccine provides enhanced cross-protection. Thus, rLS/GMCSF coexpressing the Se of regionally relevant IBV serotypes could be used in combination with live Mass to protect against regionally circulating IBV variant strains.

Protección incrementada por el virus recombinante de la enfermedad de Newcastle que expresa el ectodominio de la espícula del virus de la bronquitis infecciosa y el factor estimulante de colonias de granulocitos y macrófagos del pollo. Anteriormente se reportó que la cepa LaSota recombinante del virus de la enfermedad de Newcastle (rLS) que expresa el ectodominio de la espícula trimérica (S) de tipo Arkansas (Ark) del virus de la bronquitis infecciosa (IBV) (Se; rLS/ArkSe) proporciona una protección subóptima contra la exposición al virus de la bronquitis infecciosa. Ahora se ha desarrollado hemos desarrollado una cepa LaSota recombinante (rLS) que expresa el factor estimulante de colonias de granulocitos y macrófagos de pollo (GMCSF) y la espícula del virus de bronquitis Arkansas en un intento para mejorar la efectividad de la vacuna. En el estudio actual, primero se comparó la protección conferida por la vacunación con los virus rLS/ArkSe y rLS/ArkSe.GMCSF. Los pollos vacunados se desafiaron con un virus Arkansas virulento y la protección se determinó mediante los signos clínicos, la carga viral y la histomorfometría de la tráquea. Los resultados mostraron que la coexpresión del factor estimulante de colonias de granulocitos y macrófagos de pollo y la espícula de la cepa recombinante LaSota redujo significativamente la carga viral traqueal y las lesiones traqueales en comparación con los pollos vacunados con el virus rLS/ArkSe. En un segundo experimento, se evaluó el incremento en la protección cruzada por una vacuna atenuada de Massachusetts (Mass) mediante la primovacunación o la vacunación de refuerzo con rLS/ArkSe.GMCSF. Los pollos vacunados fueron desafiados con el virus Arkansas y se evaluó la protección. Los resultados mostraron que la primovacunación o la vacunación de refuerzo con el virus recombinante aumentó significativamente la protección cruzada conferida por el virus Massachusetts contra el desafío virulento con el virus Arkansas. Se observaron mayores reducciones de las cargas virales en los fluidos traqueales y lagrimales en pollos primovacunadoss con rLS/ArkSe.GMCSF y con vacunación de refuerzo con Massachusetts. De manera consistente, los niveles de anticuerpos Ark Se medidos con placas de ELISA recubiertas con proteína Ark Se recombinante a los 14 días después del refuerzo fueron significativamente más altos en estos pollos. De manera inesperada, el esquema de vacunación inverso, es decir, la primovacunación con Massachusetts y el refuerzo con la vacuna recombinante, resultó menos efectivo. Se concluye que una estrategia de primovacunación y refuerzo mediante el uso de rLS/ArkSe.GMCSF y la vacuna atenuada con Massachusetts usada en todo el mundo proporciona una protección cruzada aumentada. Por tanto, el virus rLS/GMCSF que coexpresa la proteína de la espícula de los serotipos regionales relevantes de bronquitis infecciosa podría usarse en combinación con una vacuna viva Massachusetts para proteger contra cepas variantes del virus de la bronquitis infecciosa que circulan regionalmente.

Limited Protection Conferred by Recombinant Newcastle Disease Virus Expressing Infectious Bronchitis Spike Protein.

Recombinant Newcastle disease virus (NDV) LaSota (LS) expressing secreted trimeric spike (S)-ectodomain (Se) of infectious bronchitis virus (IBV) (rLS/IBV.Se) was developed and evaluated for protection conferred against IBV challenge. The IBV S-ectodomain protein, which is S excluding the transmembrane anchor and short cytoplasmic domain of S2, expressed from recombinant LS corresponds to an Arkansas (Ark)-type IBV. In a first experiment, chickens were primed at 1 day of age or primed at 1 day of age and boosted at 14 days of age with 104 50% embryo infectious doses (EID50)/bird of rLS/IBV.Se and challenged with a virulent Ark strain. A single vaccination proved completely ineffective at protecting chickens against challenge, whereas priming and boosting reduced clinical signs and tracheal lesions but did not reduce viral load in lachrymal fluids. In experiment 2, the vaccine dose was increased to 107 EID50/bird and a different virulent Ark strain was used for challenge. In addition, chickens were singly immunized on either day 1 or day 10 after hatch. NDV antibody levels detected in vaccinated chickens were moderate, with hemagglutination inhibition titers varying between 4 and 5 log2. Slightly higher antibody levels to NDV were observed in chickens vaccinated on day 10 versus day 1 but without the difference achieving statistical significance. In contrast, antibody responses measured using recombinant IBV S1 protein-coated ELISA plates were significantly greater in chickens vaccinated on day 10 than on day 1. The use of a higher rLS/IBV.Se dose substantially enhanced the success of a single vaccination compared to experiment 1. Signs and tracheal lesions were reduced more effectively in chickens vaccinated at day 10 after hatch. However, as in experiment 1, vaccination did not reduce the viral loads in tear fluids of challenged chickens. Similar results, in which no reduction in viral load in the trachea was apparent from rLS/IBV.S vaccination, have been obtained by others. Further work is needed to understand the immune responses induced by this recombinant virus that seems to provide some protection against the disease but does not reduce viral loads in the upper respiratory tract.

Protección limitada conferida por un virus recombinante de la enfermedad de Newcastle que expresa la proteína de la espícula del virus de la bronquitis infecciosa. Un virus de la enfermedad de Newcastle (NDV) LaSota (LS) recombinante que expresa el ectodominio de la proteína de la espícula (S) trimérica en forma secretoria (Se) del virus de la bronquitis infecciosa (IBV) (rLS/IBV.Se) fue desarrollado y evaluado para la protección contra el desafío con el virus de la bronquitis infecciosa. El ectodominio de la proteína S del virus de la bronquitis infecciosa, que consiste en la proteína S correspondiente a la cepa Arkansas, excluyendo su anclaje transmembranario y el dominio citoplasmático corto de la proteína S2 y expresada por una cepa LaSota recombinante. En un primer experimento, los pollos fueron primovacunados al primer día de edad, o primovacunados al primer día y con un refuerzo a los 14 días de edad con 104 dosis infecciosas de embriones de pollo 50% (EID50) por ave del virus recombinante rLS/IBV.Se y desafiados con un virus virulento cepa Arkansas. Una sola vacunación demostró ser completamente ineficaz para proteger a los pollos contra el desafío, mientras que la primovacunación y el refuerzo redujeron los signos clínicos y las lesiones traqueales, pero no redujeron la carga viral en los fluidos lagrimales. En el experimento 2, la dosis de la vacuna se incrementó a 107 EID50/ave y se usó una cepa Arkansas virulenta diferente para el desafío. Además, los pollos se inmunizaron individualmente al primer día de edad o al día 10 después de la eclosión. Los niveles de anticuerpos contra el virus de Newcastle detectados en pollos vacunados fueron moderados, con títulos de inhibición de la hemaglutinación que variaron entre 4 y 5 log2. Se observaron niveles de anticuerpos ligeramente superiores contra el virus de Newcastle en pollos vacunados el día 10 en comparación con los vacunados al primer día, pero sin que la diferencia alcanzara diferencia estadística significativa. En contraste, las respuestas de anticuerpos usando placas ELISA recubiertas con proteína recombinante S1 del virus de la bronquitis infecciosa fueron significativamente mayores en pollos vacunados en el día 10 en comparación con los pollos vacunados en el primer día. Con el uso de una dosis más alta del virus rLS/IBV.Se se aumentó sustancialmente el éxito de una vacunación única en comparación con el experimento 1. Los signos y las lesiones traqueales se redujeron de manera más efectiva en los pollos vacunados en el día 10 después de la eclosión. Sin embargo, como en el experimento 1, la vacunación no redujo las cargas virales en los fluidos lagrimales de los pollos desafiados. Resultados similares, en los que no se observó una reducción en la carga viral en la tráquea por la vacunación con rLS/IBV.Se se han obtenido por otros investigadores. Se requiere de más investigación para comprender las respuestas inmunes inducidas por este virus recombinante que parece proporcionar cierta protección contra la enfermedad, pero no reduce las cargas virales en el tracto respiratorio superior.

Infectious Bronchitis Virus Population Structure Defines Immune Response and Protection.

A commercial Arkansas (Ark) Delmarva Poultry Industry (DPI)-type vaccine and a more homogeneous population of that vaccine obtained previously through adaptation to chicken embryo kidney (CEK) cells (CEK-ArkDPI) were used as a model to further understand the impact of population genetic structure on generation of immune responses and protection. In a first experiment, vaccinated chickens were challenged with an IBV Ark99-type virulent strain (AL/4614/98). Despite extensive sequence similarity between the vaccines, the more heterogeneous commercial ArkDPI was more efficient at reducing viral loads in challenged chickens, while respiratory signs and tracheal lesions were reduced similarly by either vaccine. A distinct subpopulation of the Ark challenge virus showing asparagine at S1 position 56 was consistently negatively selected by immune pressure originating from vaccination with either vaccine. Antibody levels and antibody avidity to Ark-type S1 protein were greater in CEK-ArkDPI-vaccinated chickens compared to chickens vaccinated with the more diverse commercial ArkDPI vaccine. Synchronous replication of a homogeneous virus population likely elicits clonal expansion and affinity maturation of a greater number of responding B cells compared to a diverse virus population continuously changing its proportion of phenotypes during replication. The results of a second experiment showed that during initial vaccine virus replication (24 and 48 hr postvaccination), the virus population showing increased diversity (commercial ArkDPI) achieved higher concentrations of IBV RNA in the trachea compared to the more homogenous virus. mRNA expression of genes associated with innate immune responses in the trachea 48 hr postvaccination generally showed greater upregulation in chickens vaccinated with the heterogeneous commercial ArkDPI vaccine compared to the CEK-adapted virus. The greater upregulation of these genes is likely associated with higher virus replication achieved by the heterogeneous commercial vaccine. Thus, while the adaptive antibody response was favored by the more homogenous structure of the CEK-ArkDPI vaccine population (higher antibody levels and antibody avidity), the innate immune response was favored by the more diverse viral population of the commercial ArkDPI. We confirmed previous results that distinct subpopulations in wild Ark challenge virus become selected by immune pressure originating from vaccination, and we concluded that the population structure of IBV vaccines impacts innate immune response, antibody avidity, and protection.

La estructura poblacional del virus de la bronquitis infecciosa define la respuesta inmune y la protección. Se utilizaron una vacuna comercial del tipo Arkansas (Ark) Industria Avícola de Delmarva (Delmarva Poultry Industry: DPI) y una población más homogénea de esa vacuna obtenida previamente a través de su adaptación a las células de riñón embrionario de pollo (CEK) (CEK-ArkDPI) como modelos para comprender mejor el impacto de la estructura genética de la población en la generación de respuestas inmunes y protección. En un primer experimento, los pollos vacunados fueron desafiados con una cepa virulenta de tipo Ark99 del virus de la bronquitis infecciosa (AL/4614/98). A pesar de la extensa similitud en las secuencias entre las vacunas, la cepa comercial Arkansas DPI más heterogénea fue más eficiente en la reducción de las cargas virales en los pollos desafiados, mientras que los signos respiratorios y las lesiones traqueales se redujeron de manera similar con cualquiera de las dos vacunas. Una subpoblación distinta del virus de desafío Arkansas que muestra asparagina en la posición 56 de la proteína S1 fue seleccionada de forma negativa consistentemente por la presión inmunitaria originada por la vacunación con cualquiera de las dos vacunas. Los niveles de anticuerpos y su avidez hacia la proteína S1 tipo Arkansas fueron mayores en los pollos inmunizados con la vacuna CEK-ArkDPI en comparación con los pollos vacunados con la vacuna comercial Arkansas DPI más diversa. La replicación sincrónica de una población de virus homogénea probablemente provoca la expansión clonal y la maduración por afinidad de un mayor número de células B con capacidad de respuesta en comparación con una población de virus diversa que cambia continuamente su proporción de fenotipos durante la replicación. Los resultados de un segundo experimento mostraron que durante la replicación inicial del virus de la vacuna (24 y 48 horas después de la vacunación), la población de virus que mostró una mayor diversidad (Arkansas DPI comercial) alcanzó mayores concentraciones de ARN del virus de bronquitis infecciosa en la tráquea en comparación con el virus más homogéneo. La expresión de ARNm de genes asociados con respuestas inmunes innatas en la tráquea 48 h después de la vacunación generalmente mostró una mayor regulación positiva en pollos vacunados con la vacuna comercial heterogénea Arkansas DPI en comparación con el virus adaptado a células de riñón embrionario de pollo. Es probable que la mayor regulación positiva de estos genes esté asociada con una mayor replicación del virus lograda por la vacuna comercial heterogénea. Por lo tanto, mientras que la respuesta de anticuerpos adaptativos fue favorecida por la estructura más homogénea de la población de vacunas CEK-ArkDPI (mayores niveles de anticuerpos y mayor avidez de anticuerpos), la respuesta inmune innata fue favorecida por la población viral más diversa de la vacuna Arkansas DPI comercial. Se confirman resultados previos de que distintas subpoblaciones en el virus de desafío Arkansas de tipo silvestre se seleccionan por la presión inmune originada por la vacunación y se concluye que la estructura de la población de las vacunas contra el virus de la bronquitis infecciosa afecta la respuesta inmune innata, la avidez de los anticuerpos y la protección.

Infectious Bronchitis Virus Vaccination at Day 1 of Age Further Limits Cross Protection.

Cross-protection and immune responses elicited by infectious bronchitis virus (IBV) vaccination on Day 1 of age or at later time points were examined. Specific-pathogen-free chickens were vaccinated with a Massachusetts-type vaccine and heterologous challenge was performed with an Arkansas (Ark) -type virulent strain. In Trial 1, chickens vaccinated on Day 1 or Day 10 of age were challenged 21 days after vaccination. Analysis of tracheal histopathology and viral load demonstrated less cross protection when vaccination was performed on Day 1 of age. In Trial 2, chickens were vaccinated on Day 1 or Day 14 of age. A somewhat stronger systemic antibody response to IBV was detected in chickens vaccinated at 14 days of age. In addition, avidity of antibodies to Ark-type S1 protein elicited by vaccination at 14 days of age was greater. Differences in immune-cell populations in the Harderian gland (HG) observed at the time of sampling (35 days following vaccination) between chickens vaccinated at 1 day or 14 days of age indicated greater, rather than reduced, immune activity in the chickens vaccinated at 1 day of age. These differences are, perhaps, a result of the higher levels of persisting vaccine virus observed in the younger chickens. Both nonvaccinated/challenged groups showed significantly higher (P < 0.05) proportions of B cells and CD8+ T cells 7 days after challenge than age-matched vaccinated/challenged groups or age-matched nonvaccinated/nonchallenged control groups. These results indicate infiltration and/or expansion of B cells and CD8+ cells in HGs following challenge of nonvaccinated chickens. A fortuitous finding was that the more immature immune system of chickens vaccinated at 1 day of age was less effective at clearing vaccine virus after vaccination. Collectively, the current results indicate that IBV vaccination at 1 day of age can decrease the potential for heterologous cross protection compared with vaccination at least 10 days after hatch. A lower level of antibody affinity maturation likely contributes to decreased cross protection.

Límites en la protección cruzada inducida por la vacunación contra el virus de la bronquitis infecciosa en el primer día de edad. Se examinó la protección cruzada y la respuesta inmune inducida por la vacunación contra el virus de la bronquitis infecciosa en el primer día de edad o en posteriores puntos en el tiempo. Se vacunaron pollos libres de patógenos específicos con una vacuna de tipo Massachusetts y se realizó un desafío heterólogo con una cepa virulenta del tipo Arkansas (Ark). En el ensayo 1, pollos vacunados en el día uno o en el día 10 de edad fueron desafiados a los 21 días después de la vacunación. El análisis de la histopatología traqueal y de la carga viral demostró una menor protección cruzada cuando la vacunación se realizó en el día uno de edad. En el ensayo dos, los pollos fueron vacunados en el día uno o en el día 14 de edad. Se detectó una respuesta de anticuerpos sistémicos ligeramente más elevada contra el virus de la bronquitis infecciosa en los pollos vacunados a los 14 días de edad. Además, la avidez de los anticuerpos contra la proteína S1 tipo Arkansas inducida por la vacunación a los 14 días de edad fue mayor. Las diferencias en las poblaciones de células inmunitarias en la glándula de Harder observadas en el momento del muestreo 35 días después de la vacunación en los pollos vacunados al primer día o a los14 días de edad indicaron una mayor actividad inmunitaria en lugar de reducción en los pollos vacunados al primer día de edad, posiblemente como resultado de los niveles más altos de virus vacunal persistente observados en los pollos más jóvenes. Ambos grupos no vacunados y desafiados mostraron proporciones significativamente más altas (P <0.05) de células B y células T CD8+ siete días después del desafío en comparación con los grupos vacunados y desafiados de la misma edad o los grupos control no vacunados y no desafiados de la misma edad. Estos resultados indican la infiltración y/o expansión de las células B y de células CD8+ en la glándula de Harder después del desafío de los pollos no vacunados. Un hallazgo fortuito fue que el sistema inmune más inmaduro de los pollos vacunados al primer día de edad fue menos efectivo para eliminar el virus vacunal después de la vacunación. En conjunto, los resultados actuales indican que la vacunación con el virus de la bronquitis infecciosa al primer día de edad puede disminuir el potencial de protección cruzada heteróloga en comparación con la vacunación al menos 10 días después de la eclosión. Un nivel más bajo de maduración de la afinidad de anticuerpos probablemente contribuye a disminuir la protección cruzada.

Kidney Cell-Adapted Infectious Bronchitis Virus Arkansas Delmarva Poultry Industry Vaccine Confers Effective Protection Against Challenge.

We previously demonstrated that adaptation of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas Delmarva Poultry Industry (ArkDPI)-derived vaccine to chicken embryo kidney (CEK) cells shifted the virus population towards homogeneity in spike (S) and nonstructural protein genes. Moreover, the typical Ark vaccine subpopulations emerging in chickens vaccinated with commercial Ark vaccines were not detected in chickens vaccinated with the CEK-adapted virus. In this study, chickens vaccinated with a low dose (1.6 × 10(3) EID50/bird, where EID50 is 50% embryo infectious dose) of CEK-adapted Ark vaccine at 5 days of age showed a significant reduction of IBV RNA in lachrymal fluids and decreased incidence of IBV RNA detection in tracheal swabs 5 days after challenge compared to unvaccinated challenged chickens. In a second experiment, 5-day-old chickens were vaccinated with 10(4) or 10(5) EID50/chicken of CEK-adapted Ark vaccine, and protection was compared to chickens vaccinated with 10(5) EID50/chicken of the commercial ArkDPI-derived vaccine from which the CEK-adapted virus originated. All vaccinated chicken groups showed a significant reduction of respiratory signs and viral load 5 days after Ark virulent challenge compared to unvaccinated challenged controls. No viral subpopulations different from the challenge virus were detected in chickens vaccinated with CEK-Ark after challenge. In contrast, IBV S1 sequences differing from the predominant population in the challenge virus were detected in several chickens vaccinated with the commercial Ark attenuated vaccine. From an applied perspective, the CEK-adapted IBV ArkDPI-derived vaccine is an improved and effective vaccine candidate with which to protect chickens against virulent Ark-type strains.

Cross-Protection by Infectious Bronchitis Viruses Under Controlled Experimental Conditions.

Infectious bronchitis virus (IBV) cross-protection trials were performed in healthy chickens maintained under controlled environmental conditions. Chickens primed or primed and boosted with a Massachusetts (Mass)-type attenuated vaccine were subsequently challenged with either IBV Arkansas (Ark) or GA13-type virulent strains. In addition, Ark-vaccinated chickens were challenged with IBV GA13. Spike protein 1 (S1) amino acid identities between IBV vaccine and challenge strains varied from 76.0% to 77.3%. Contrary to expectations, assessments of clinical signs, viral load, and histopathology indicated a significant level of cross-protection among these antigenically distant IBV strains. Moreover, prime and booster vaccination with Mass protected against GA13 and improved protection against Ark when compared with Mass single vaccination. These results emphasize the need to include both single vaccination control groups and control groups primed and boosted with a single serotype when testing the efficacy of IBV protectotypes and/or novel IBV vaccine combinations against heterologous serotypes under controlled experimental conditions. Such controls are of distinct importance in experiments supporting the introduction of attenuated IBV vaccine strains exotic to regions, since these exotic strains may provide new genetic material for recombination and emergence of novel IBV strains.

Evaluation of capillary and myofiber density in the pectoralis major muscles of rapidly growing, high-yield broiler chickens during increased heat stress.

Skeletal muscle development proceeds from early embryogenesis through marketing age in broiler chickens. While myofiber formation is essentially complete at hatching, myofiber hypertrophy can increase after hatch by assimilation of satellite cell nuclei into myofibers. As the diameter of the myofibers increases, capillary density peripheral to the myofiber is marginalized, limiting oxygen supply and subsequent diffusion into the myofiber, inducing microischemia. The superficial and deep pectoralis muscles constitute 25% of the total body weight in a market-age bird; thus compromise of those muscle groups can have profound economic impact on broiler production. We hypothesized that marginal capillary support relative to the hypertrophic myofibers increases the incidence of microischemia, especially in contemporary high-yield broilers under stressing conditions such as high environmental temperatures. We evaluated the following parameters in four different broiler strains at 39 and 53 days of age when reared under thermoneutral (20 to 25 C) versus hot (30 to 35 C) environmental conditions: capillary density, myofiber density and diameter, and degree of myodegeneration. Our data demonstrate that myofiber diameter significantly increased with age (P > or = 0.0001), while the absolute numbers of capillaries, blood vessels, and myofibers visible in five 400 x microscopic fields decreased (P > or = 0.0001). This is concomitant with marginalization of vascular support in rapidly growing myofibers. The myofiber diameter was significantly lower with hot environmental temperatures (P > or = 0.001); therefore, the absolute number of myofibers visible in five 400X microscopic fields was significantly higher. The incidence and subjective degree of myodegeneration characterized by loss of cross-striations, myocyte hyperrefractility, sarcoplasmic vacuolation, and nuclear pyknosis or loss also increased in hot conditions. Differences among strains were not observed.

S1 of distinct IBV population expressed from recombinant adenovirus confers protection against challenge.

Protective properties of three distinct infectious bronchitis virus (IBV) Ark Delmarva poultry industry (ArkDPI) S1 proteins encoded from replication-defective recombinant adenovirus vectors were investigated. Using a suboptimal dose of each recombinant virus, we demonstrated that IBV S1 amino acid sequences showing > or = 95.8% amino acid identity to the S1 of the challenge strain differed in their ability at conferring protection. Indeed, the S1 sequence of the IBV population previously designated C4 (AdIBVS1.C4), which protected the most poorly, differs from the S1 sequence of population C2 (AdIBVS1.C2), which provided the highest protection, only at amino acid position 56. The fact that a change in one amino acid in this region significantly altered the induction of a protective immune response against this protein provides evidence that the first portion of S1 displays relevant immunoprotective epitopes. Use of an optimal dose of AdIBVS1.C2 effectively protected chickens from clinical signs and significantly reduced viral load after IBV Ark virulent challenge. Moreover, increased numbers of both IgA and IgG IBV-specific antibody secreting lymphocytes were detected in the spleen after challenge. The increased response detected for both IgA and IgG lymphocytes after challenge might be explained by vaccine-induced B memory cells. The fact that a single vaccination with Ad/IBVS1.C2 provides protection against IBV challenge is promising, because Ad-vectored vaccines can be mass delivered by in ovo inoculation using automated in ovo injectors.

Multicentric cutaneous neuroendocrine (Merkel cell) carcinoma in a dog.

Multicentric cutaneous neuroendocrine (Merkel cell) carcinoma was diagnosed in a 5-year-old castrated male Keeshond dog with multiple firm nodular cutaneous masses. The neoplastic tissue locally effaced the periadnexal and deep dermis and consisted of densely cellular confluent clusters of round to polygonal cells supported by a delicate fibrovascular stroma. The cells were moderately immunoreactive with chromogranin A, synaptophysin, and cytokeratin. Ultrastructurally, the cells had characteristic membrane-bound dense-core neuroendocrine granules approximately 120 nm in diameter and randomly dispersed throughout the cytoplasm. Effacement of dermal structures and multicentric distribution suggested low-grade malignant phenotype. These findings contrast with the typical benign behavior of canine cutaneous neuroendocrine tumors.

Pathogenesis of infectious bronchitis virus in vaccinated chickens of two different major histocompatibility B complex genotypes.

Infectious bronchitis (IB) disease progression in vaccinated chickens after challenge was evaluated in a single commercial line of layer chickens presenting two different major histocompatibility complex (MHC) B complex genotypes. MHC B genotypes were determined by DNA sequence-based typing of BF2 alleles. In total, 33 B2/B15 and 47 B2/B21 chickens were vaccinated with an Ark-type IB virus (IBV) attenuated vaccine and challenged with Ark-type IBV field isolate AL/4614/98 14 days later. Additional chickens of both genotypes served as unvaccinated/challenged and unvaccinated/nonchallenged controls. Clinical signs, histopathologic analysis, detection of IBV genomes in tears, and IBV-specific immunoglobulin A (IgA) in tears were used to evaluate disease progression and immune response. The incidence of IBV respiratory signs was significantly higher in B2/21 than in B2/B15 MHC genotype birds. However, neither the severity and duration of respiratory signs nor the severity and incidence of histologic lesions differed significantly with MHC genotype. The levels of IBV-specific IgA in tears of vaccinated and challenged chickens did not differ significantly between MHC genotypes. IBV genomes were present in the tears of vaccinated and challenged birds, and the incidence of detectable IBV genomes did not vary significantly with MHC B genotype. From an applied perspective, these results indicate that vaccinated commercial outbred chickens with these MHC genotypes are equally resistant to IBV.

Toxoplasma gondii infection induces apoptosis in noninfected macrophages: role of nitric oxide and other soluble factors.

Apoptosis has been found to help in the defence against pathogens. Infection with the obligate intracellular parasite Toxoplasma gondii is known to trigger host-cell apoptosis. When using a T. gondii-infected macrophage cell line, J774A.1, treatment with IFN-gamma significantly enhanced apoptosis in noninfected bystander cells while parasitized cells became relatively resistant. Infection and IFN-gamma treatment activated the expression of inducible nitric oxide synthase (iNOS), and the production of nitric oxide (NO) and treatment of cells with an iNOS inhibitor, N(G)-monomethlyl-L-arginine acetate (L-NMMA) reduced the apoptosis frequency. However, the reversal was only partial suggesting that not only NO, but also other, as of yet, unknown factors are induced. Finally, we studied the effect in vivo by infecting mice with either a virulent or an avirulent strain. Challenge with the virulent strain lead to a higher parasite burden, induced host-cell apoptosis in peritoneal cells, and produced higher levels of IFN-gamma and NO. Moreover, treatment of mice with a NO synthase inhibitor, aminoguanidine, partially inhibited the host-cell apoptosis induced by the parasite infection. Altogether, our findings indicate that apoptosis in bystander host cells is due to the secretion of NO and other soluble factors released by parasite-infected cells.

Hemangiopericytoma in the eyelid of a horse.

Hemangiopericytoma (HP) is a well-recognized neoplasm arising from vascular pericytes that has been reported only in the dog and man. In this study, we describe a 14-year-old female Arabian horse that was presented for surgical excision of a 2-cm-diameter expansile subcuticular mass in the right lower eyelid. Histologically, the mass consisted of loosely arranged interlacing streams and storiform bundles of spindle cells that often formed distinct whorls around a central capillary and bundles of collagen (Antoni A-like pattern). Immunohistochemical analysis revealed strong diffuse cytoplasmic immunoreactivity for vimentin and focal immunoreactivity for smooth muscle actin, whereas neoplastic cells did not stain for Factor VIII-related antigen, Glial fibrillary acidic protein (GFAP), or S100. On the basis of histomorphology and immunohistochemical reactivity, the present tumor was diagnosed as HP. To our knowledge, this is the first report describing a HP in a horse.

The avian major histocompatibility complex influences bacterial skeletal disease in broiler breeder chickens.

This study evaluated bacterial skeletal disease in conjunction with the major histocompatibility complex (MHC) in a genetically pure line of broiler breeder chickens. Chickens from six broiler breeder flocks were examined for skeletal lesions, bacterial pathogens, and MHC genotype. During a 10-week period, eighty-eight, 9- to 21-week-old lame chickens and 34 normal, age-matched controls were selected. Tenosynovitis, arthritis, and femoral or tibiotarsal (or both) osteomyelitis occurred in 86 of 88 (97.7%) lame chickens. Ninety-five bacterial isolates were obtained from 83 of 88 (94.3%) lame birds and 4 of 34 (11.8%) controls. Staphylococcus spp. was isolated from 72.6% of the skeletal lesions, predominantly Staphylococcus aureus (38.9%). MHC B complex genotypes were determined by hemagglutination for 88 lame birds, 34 controls, and 200 randomly selected birds from each of the six flocks (1,200 total). Combined chi-square analysis revealed that the homozygous MHC genotypes B(A4/A4) (chi(2) = 14.54, P = 0.0063) and B(A12/A12) (chi(2) = 42.77, P = 0.0001) were overrepresented in the sample of symptomatic birds compared with random samples from the same flocks. The homozygous A4 and A12 MHC genotypes influenced flock chi-square values more than the corresponding heterozygotes. An MHC B complex influence on bacterial skeletal disease was apparent in this line of broiler breeders.

Oral infection with chicken anemia virus in 4-wk broiler breeders: lack of effect of major histocompatibility B complex genotype.

The pathologic consequences of chicken anemia virus (CAV) oral inoculation in 4-wk-old broiler breeders of different major histocompatibility B complex (MHC) genotypes were evaluated. MHC B complex was determined by hemagglutination and sequence-based typing. Clinical signs, serology, gross lesions, histopathologic analysis, and CAV genome quantification were used to evaluate disease progression. Clinical disease was not apparent in the inoculated broilers throughout the experimental period. At 14 days postinoculation, antibodies against CAV were detected in 26.4% (29/110) of the inoculated birds. The distribution of percent positive was 34.6% (9/26) and 32.3% (10/31) of the chickens with B A9/A9 and B A9/A4 MHC genotypes, respectively, and seroconversion in six other genotypes was 19% (10/53). These differences among MHC genotypes for specific seroconversion rate were not statistically significant. CAV genomes were detected in the thymus of 87.7% (93/110) of the inoculated birds with no statistically significant differences between MHC genotypes. Mild thymic lymphocytolysis, lymphedema, and medullary hemorrhage were observed in the inoculated chickens. Histomorphometric analysis showed that cortical lymphocyte-to-parenchyma ratios did not differ between inoculated and uninoculated groups or among MHC genotypes. Similar findings have been reported previously in white-leghorn chickens of similar age, suggesting that broilers show a similar resistance to the effects of CAV infection at this age. The absence of significant clinical and pathological changes in the orally inoculated broilers at this age contrasts with CAV-associated thymus damage seen frequently in condemned commercial broilers at harvest.

Pathogenesis of chicken anemia virus: comparison of the oral and the intramuscular routes of infection.

The events during the pathogenesis of chicken anemia virus (CAV) infection following intramuscular (IM) and oral inoculation were further elucidated and compared by sequential clinical, pathologic, and morphometric histopathologic evaluations, and by sequential determination of CAV genome concentrations in different organs. Specific-pathogen-free chickens were inoculated by IM or oral routes with the same dose (2 x 10(6) mean tissue culture infective dose [TCID50]) of CAV isolate 03-4876 at 1 day of age. Weights and hematocrits were obtained at 7, 10, 14, 18, 21, 25, and 28 days postinoculation (DPI). Seven birds from each group were necropsied at 7, 10, 14, and 28 DPI, and samples of thymus, Harderian gland, and cecal tonsils (CT) were obtained for histopathologic examination and CAV genome quantification by real-time polymerase chain reaction. Peak CAV genome concentrations were detected in the thymus at 10 and 14 DPI in the IM and orally infected chickens, respectively. High CAV DNA concentrations were maintained throughout the experimental period until 28 DPI, despite specific seroconversion occurring by 14 DPI in the IM-inoculated chickens. CAV was isolated from both orally and IM-infected chickens 28 DPI. Peak CAV genomes in the thymuses of IM and orally infected chickens coincided with peak lymphocyte depletion in these organs. Lymphocyte repopulation of the thymus occurred by 28 DPI in spite of the presence of the virus in the organs of both infected chicken groups. CAV genomes were detected in the CT, but histopathologic changes were not observed. Compared with the IM route of infection, orally infected chickens did not show apparent signs of illness. Clinical parameters, including reduction of weight gains and hematocrits, and gross and histopathologic changes were delayed and less severe in the orally inoculated chickens. This was concurrent with a delay in accumulation of CAV genomes in the thymus of these chickens.

Disseminated toxoplasmosis in a captive ring-tailed lemur (Lemur catta).

A 3-yr-old secundiparous female ring-tailed lemur presented to the Auburn University Small Animal Clinic with signs of dyspnea, lethargy, and anorexia. The animal died before she could be examined, and a full necropsy was immediately performed. Provisional necropsy findings included moderate pneumonia and hepatopathy. Acute interstitial pneumonia and focal hepatocellular necrosis were confirmed histologically. Lung impression smears, histopathology, electron microscopy, immunohistochemistry, and tissue culture isolation resulted in a diagnosis of acute disseminated Toxoplasma gondii infection, which was confirmed by polymerase chain reaction. The isolate of T. gondii was avirulent for mice and was named AU Tgl and genetically is type II. The source of the infection remains unclear, but speculation suggests contaminated fruit or blackbirds (Passeriformes: Icteridae) acting as transport hosts for oocysts from nondomestic felids and feral cats on the property.

Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii.

A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms. Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution. Significant progress in studying the cell biology of this parasite has been accomplished over the last few years. Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T. gondii. In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.

The Toxoplasma gondii protein ROP2 mediates host organelle association with the parasitophorous vacuole membrane.

Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH(2)-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30-amino acid (aa) NH(2)-terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH(2)-terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.

Toxoplasma gondii ADP-ribosylation factor 1 mediates enhanced release of constitutively secreted dense granule proteins.

Toxoplasma gondii dense granules are morphologically similar to dense matrix granules in specialized secretory cells, yet are secreted in a constitutive, calcium-independent fashion. We previously demonstrated that secretion of dense granule proteins in permeabilized parasites was augmented by the non-hydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (Chaturvedi, S., Qi, H., Coleman, D. L., Hanson, P., Rodriguez, A., and Joiner, K. A. (1998) J. Biol. Chem. 274, 2424-2431). As now demonstrated by pharmacological and electron microscopic approaches, GTPgammaS enhanced release of dense granule proteins in the permeabilized cell system. To investigate the role of ADP-ribosylation factor 1 (ARF1) in this process, a cDNA encoding T. gondii ARF1 (TgARF1) was isolated. Endogenous and transgenic TgARF1 localized to the Golgi of T. gondii, but not to dense granules. An epitope-tagged mutant of TgARF1 predicted to be impaired in GTP hydrolysis (Q71L) partially dispersed the Golgi signal, with localization to scattered vesicles, whereas a mutant impaired in nucleotide binding (T31N) was cytosolic in location. Both mutants caused partial dispersion of a Golgi/trans-Golgi network marker. TgARF1 mutants inhibited delivery of the secretory reporter, Escherichia coli alkaline phosphatase, to dense granules, precluding an in vivo assessment of the role of TgARF1 in release of intact dense granules. To circumvent this limitation, recombinant TgARF1 was purified using two separate approaches, and used in the permeabilized cell assay. TgARF1 protein purified on a Cibacron G3 column and able to bind GTP stimulated dense granule secretion in the permeabilized cell secretion assay. These results are the first to show that ARF1 can augment release of constitutively secreted vesicles at the target membrane.