Rodrigo A. Gallardo

Novel mechanisms revealed in the trachea transcriptome of resistant and susceptible chicken lines following infection with Newcastle disease virus

ABSTRACT Newcastle disease virus (NDV) has a devastating impact on poultry production in developing countries. This study examined the transcriptome of tracheal epithelial cells from two inbred chicken lines that differ in NDV susceptibility after challenge with a high-titer inoculum of lentogenic NDV. The Fayoumi line had a significantly lower NDV load postchallenge than the Leghorn line, demonstrating the Fayoumi lines classification as a relatively NDV-resistant breed. Examination of the trachea transcriptome showed a large increase in immune cell infiltration in the trachea in both lines at all times postinfection. The pathways conserved across lines and at all three time points postinfection included iCOS-iCOSL signaling in T helper cells, NF-κB signaling, the role of nuclear factor of activated T cells in the regulation of the immune response, calcium-induced T lymphocyte apoptosis, phospholipase C signaling, and CD28 signaling in T helper cells. Although shared pathways were seen in the Fayoumi and Leghorn lines, each line showed unique responses as well. The downregulation of collagen and the activation of eukaryotic translation initiation factor 2 signaling in the Fayoumis relative to the Leghorns at 2 days postinfection may contribute to the resistance phenotype seen in the Fayoumis. This study provides a further understanding of host-pathogen interactions which could improve vaccine efficacy and, in combination with genome-wide association studies, has the potential to advance strategies for breeding chickens with enhanced resistance to NDV.

Bovine viral diarrhea virus fetal persistent infection after immunization with a contaminated modified-live virus vaccine.

The objective was to determine whether a multivalent modified-live virus vaccine containing noncytopathic bovine viral diarrhea virus (BVDV) administered off-label to pregnant cattle can result in persistently infected fetuses and to assess whether vaccinal strains can be shed to unvaccinated pregnant cattle commingling with vaccinates. Nineteen BVDV-naïve pregnant heifers were randomly assigned to two groups: cattle vaccinated near Day 77 of gestation with modified-live virus vaccine containing BVDV-1a (WRL strain), bovine herpes virus-1, parainfluenza 3, and bovine respiratory syncytial virus (Vx group; N = 10) or control unvaccinated cattle (N = 9). During the course of the study a voluntary stop-sale/recall was conducted by the manufacturer because of the presence of a BVDV contaminant in the vaccine. At Day 175 of gestation, fetuses were removed by Cesarean section and fetal tissues were submitted for virus isolation, and quantitative reverse transcription polymerase chain reaction using BVDV-1- and BVDV-2-specific probes. Nucleotide sequencing of viral RNA was performed for quantitative reverse transcription polymerase chain reaction-positive samples. Two vaccinated and two control heifers aborted their pregnancies, but their fetuses were unavailable for BVDV testing. Virus was isolated from all eight fetuses in the Vx group heifers and from 2 of 7 fetuses in the control unvaccinated heifers. Only BVDV-2 was detected in fetuses from the Vx group, and only BVDV-1 was detected in the two fetuses from the control group. Both BVDV-1 and BVDV-2 were detected in the vaccine. In conclusion, vaccination of pregnant heifers with a contaminated modified-live BVDV vaccine resulted in development of BVDV-2 persistently infected fetuses in all tested vaccinated animals. Furthermore, BVDV was apparently shed to unvaccinated heifers causing fetal infections from which only BVDV-1 was detected.

Immunoglobulin A as an Early Humoral Responder After Mucosal Avian Coronavirus Vaccination

SUMMARY Infectious bronchitis virus (IBV) is a highly contagious coronavirus prevalent in all countries with an extensive poultry industry and continues to cause economic losses. IBV strains of the Ark serotype are highly prevalent in the Southeastern United States despite extensive vaccination. One explanation for this observation is the high genetic variability of IBV. In addition, IBV Ark-type vaccines may induce suboptimal mucosal immune responses, contributing to the prevalence and persistence of the Ark serotype. To test this hypothesis, chickens were ocularly vaccinated with a commercially available live attenuated IBV Ark–Delmarva Poultry Industry vaccine strain and both mucosal and systemic antibody responses were measured. The highest immunoglobulin A (IgA) spot-forming cell (SFC) response was observed in the Harderian glands (HG) and to a lesser extent in the spleen and conjunctiva-associated lymphoid tissues, while a limited IgG SFC response was observed in either the mucosal or systemic immune compartment. Interestingly, the peak IgA SFC response occurred 2 days earlier in spleen than in the head-associated lymphoid tissues despite ocular vaccination. Furthermore, IgA IBV-specific antibody levels significantly increased over controls 3 days earlier in tears and 4 days earlier in plasma than did IgG antibodies. IgA antibody levels were higher than IgG antibody levels throughout the primary response in tears and were similar in magnitude in plasma. In addition, a very early increase in IgA antibodies on day 3 postvaccination was observed in tears; such a response was not observed in plasma. This early increase is consistent with a mucosal T-independent IgA response to IBV. In the secondary response the IBV antibody levels significantly increased over controls starting on day 1 after boosting, and the IgG antibody levels were higher than the IgA antibody levels in both tears and plasma. In summary, ocular vaccination induced higher IgA antibodies in the primary IBV response, while the memory response is dominated by IgG antibodies. Thus, lower mucosal IgA antibody levels are observed upon secondary exposure to IBV, which may contribute to vulnerability of host epithelial cells to infection by IBV and persistence of the Ark serotype. RESUMEN La inmunoglobulina A como una respuesta humoral temprana después de la vacunación en las mucosas con coronavirus aviar. El virus de la bronquitis infecciosa (IBV) es un coronavirus muy contagioso prevalente en todos los países con una industria avícola extensa y sigue causando pérdidas económicas. Las cepas del virus de bronquitis del serotipo Arkansas son altamente prevalentes en el sureste de los Estados Unidos a pesar de la vacunación extensiva. Una explicación de esta observación es la alta variabilidad genética del virus de bronquitis. Además, las vacunas del tipo Arkansas pueden inducir respuestas inmunitarias subóptimas en las mucosas, que contribuyen a la prevalencia y persistencia del serotipo Arkansas. Para comprobar esta hipótesis, los pollos fueron vacunados ocularmente con una cepa vacunal viva atenuada disponible comercialmente del serotipo Arkansas DPI (Delmarva Poultry Industry) y las respuestas inmunitarias de las mucosas y de anticuerpos sistémicos fueron medidas. El sitio de células formadoras de inmunoglobulina A con mayor producción se observó en la glándula de Harder y en menor medida en el bazo y en los tejidos linfoides asociados a la conjuntiva, mientras que se observó una limitada respuesta de los sitios de células formadoras de IgG, ya sea en la mucosa o en el compartimento inmune sistémico. De manera interesante, el pico de respuesta de los sitios celulares formadores de IgA se produjo dos días antes en el bazo que en los tejidos linfoides asociados con la cabeza a pesar de que la vacunación fue por vía ocular. Por otra parte, los niveles de anticuerpos IgA específicos contra el virus de bronquitis aumentaron significativamente en los controles tres días antes en lágrimas y cuatro días antes en el plasma en comparación con los anticuerpos IgG. Los niveles de anticuerpos IgA fueron más altos que los niveles de anticuerpos de IgG a lo largo de la respuesta primaria en las lágrimas y fueron similares en magnitud en el plasma. Además, un aumento muy temprano en la IgA en el día tres después de la vacunación se observó en las lágrimas; dicha respuesta no fue observada en el plasma. Este aumento precoz es consistente con una respuesta de IgA T-independiente en la mucosa contra el virus de bronquitis. En la respuesta secundaria, los niveles de anticuerpos contra el virus de la bronquitis aumentaron significativamente en los controles a partir del día uno después de la vacunación de refuerzo y los niveles de anticuerpos IgG fueron más altos que los niveles de anticuerpos IgA tanto en lágrimas como en el plasma. En resumen, la vacunación ocular induce niveles de anticuerpos IgA más altos en la respuesta primaria contra el virus de bronquitis, mientras que la respuesta de memoria está dominada por anticuerpos IgG. De esta manera, niveles bajos de IgA en la mucosa se observan después de la exposición secundaria al virus de la bronquitis infecciosa, lo que puede contribuir a la vulnerabilidad de las células epiteliales del huésped a la infección por dicho virus y a la persistencia del serotipo Arkansas.

Effects of challenge with very virulent infectious bursal disease virus reassortants in commercial chickens.

SUMMARY Pathogenicity and immune responses were characterized in commercial broilers and layers challenged with very virulent infectious bursal disease virus (vvIBDV) reassortants (vvIBDV segment A + serotype 2 segment B and vvIBDV segment A + classic virulent segment B) at 7 days of age. In addition, functional immunosuppression was evaluated after challenge with infectious bronchitis virus (IBV) at 15 days of age. Layers showed higher levels and increased persistence of IBDV- and IBV-specific maternal antibodies than broilers at 1, 13, and 28 days of age. Cytokine gene expression was evaluated, after IBDV challenge, as an indicator of the innate immune function. Similar results were detected between the groups inoculated with vvIBDV reassortants. Interleukin-1&bgr; (IL-1&bgr;) in the bursa of layers demonstrated down-regulation at 1 day postinfection (DPI; 8 days of age), and no changes at 4 DPI (11 days of age) compared with controls. In broilers, IL-6 expression in the bursa was down-regulated 1 DPI (8 days of age) and up-regulated at 4 DPI (11 days of age). A significant lymphoid depletion was detected at 21 DPI (28 days of age) in broilers exposed to a reassortant of vvIBDV segment A and classic virulent IBDV segment B. Finally, reduced specific antibodies against IBV measured 13 days after challenge were detected in layer and broiler chickens inoculated with a reassortant serotype 2 IBDV in segment B, suggesting functional immunosuppression. These results provide evidence indicating that current IBDV vaccination of breeders does not completely protect progeny chickens from challenge with reassortant vvIBDV. RESUMEN Efectos en aves comerciales del desafío con virus de la enfermedad infecciosa de la bolsa muy virulentos y reacomodados genéticamente. Se caracterizaron la patogenicidad y la respuesta inmune en pollos de engorde y en aves de postura comerciales desafiados con el virus de la enfermedad infecciosa de la bolsa muy virulentos (con las siglas en Inglés vvIBDV) reacomodados genéticamente (segmento A tipo vvIBDV + segmento B del serotipo 2 y segmento A muy virulento + segmento B clásico virulento) a los 7 días de edad. Además, se evaluó la inmunodepresión funcional después del desafío con el virus de la bronquitis infecciosa (IBV) a los 15 días de edad. Las aves de postura mostraron niveles más altos y una mayor persistencia de anticuerpos maternos específicos contra el virus de Gumboro y de la bronquitis infecciosa en comparación con los pollos de engorde en los días uno, 13, y 28 días de edad. Se evaluó la expresión de genes para citoquinas después de la exposición del virus de Gumboro, como un indicador de la respuesta inmune innata. Se detectaron resultados similares entre los grupos inoculados con los virus altamente virulentos reacomodados. La interleucina-1&bgr; (IL-1&bgr;) en la bolsa de las aves de postura mostró regulación a la baja en el día uno después de la inoculación (ocho días de edad), y sin cambios a los cuatro días después de la inoculación (11 días de edad), en comparación con las aves controles. En los pollos de engorde, la expresión de IL-6 en la bolsa mostró regulación a la baja al día uno de edad (8 días de edad) y regulación a la alta a los cuatro días después de la inoculación (11 días de edad). Se detectó despoblación linfoide significativa a los 21 días postinoculación (28 días de edad) en pollos de engorde expuestos a un virus con genoma reacomodado con un segmento A de cepa muy virulenta y un segmento B de cepa clásica virulenta. Por último, la reducción de anticuerpos específicos contra el virus de la bronquitis infecciosa frente a IBV detectados a los 13 días después del desafío se detectaron en las aves de postura y de pollos de engorde pollos inoculados con un virus reacomodado serotipo 2 en el segmento B, lo que sugiere inmunodepresión funcional. Estos resultados proporcionan evidencia que indica que la vacunación actual contra el virus de Gumboro en las aves reproductoras no protege por completo a la progenie contra el desafío con virus muy virulentos reacomodados.

Diatoms and diatomaceous earth as novel poultry vaccine adjuvants.

&NA; Diatoms are single cell eukaryotic microalgae; their surface possesses a porous nanostructured silica cell wall or frustule. Diatomaceous earth (DE) or diatomite is a natural siliceous sediment of diatoms. Since silica has been proved to have adjuvant capabilities, we propose that diatoms and DE may provide an inexpensive and abundant source of adjuvant readily available to use in livestock vaccines. In a first experiment, the safety of diatoms used as an adjuvant for in‐ovo vaccination was investigated. In a second experiment, we assessed the humoral immune response after one in‐ovo vaccination with inactivated Newcastle Disease Virus (NDV) and DE as adjuvant followed by 2 subcutaneous boosters on d 21 and 29 of age. In both experiments, results were compared to Freunds incomplete adjuvant and aluminum hydroxide. No detrimental effects on hatchability and chick quality were detected after in‐ovo inoculation of diatoms and DE in experiments 1 and 2 respectively. In experiment 2 no humoral responses were detected after the in‐ovo vaccination until 29 d of age. Seven d after the second subcutaneous booster an antibody response against NDV was detected in chickens that had received vaccines adjuvanted with Freunds incomplete adjuvant, aluminum hydroxide, and DE. These responses became significantly higher 10 d after the second booster. Finally, 15 d after the second booster, the humoral responses induced by the vaccine with Freunds incomplete adjuvant were statistically higher, followed by comparable responses induced by vaccines containing DE or aluminum hydroxide that were significantly higher than DE+PBS, PBS+INDV and PBS alone. From an applied perspective, we can propose that DE can serve as a potential adjuvant for vaccines against poultry diseases.

Persistence of Highly Pathogenic and Low Pathogenic Avian Influenza Viruses in Footbaths and Poultry Manure

SUMMARY A questionnaire was designed in order to gather information about bedding material and footbath preparation and maintenance in different productive units across the state of California.This information was used to plan two experiments. In the first experiment, we tested the effectiveness of footbaths in inactivating highly pathogenic (HP) and low pathogenic (LP) avian influenza viruses (AIVs) on rubber boots. Surprisingly, quaternary ammonia– and quaternary ammonia + glutaraldehyde–based footbaths were not able to eliminate live HPAIV (H5N8) and LPAIV (H6N2) particles on boots, while a chlorine-based granulated disinfectant was able to destroy the virus at contact. These results demonstrated the potential of AIV, particularly the HPAIV isolate, to persist even if exposed to disinfecting footbaths, and suggest that footbaths, as a single tool, are not capable of preventing pathogen introduction into commercial flocks. In the second experiment, we investigated the persistence of HPAIV (H5N8) and LPAIV (H6N2) in bedding material and feces obtained from turkey, broiler, and egg-layer commercial productive units. Samples were collected at different times after spiking the bedding materials and feces. Results showed that HPAIV (H5N8) was more persistent than LPAIV (H6N2) in layer feces and bedding material obtained from commercial broilers and turkeys. Live HPAIV particles persisted 96 hr, the last time point measured, in layer feces and less than 60 hr in broiler and turkey bedding. In contrast, LPAIV persisted less than 24 hr after being spiked in all the different substrates. Further research in biosecurity practices such as footbath preparation and maintenance and better understanding of the mechanism of the increased persistence of AIV is warranted in order to identify effective litter treatments that destroy live virus in bedding material.

Novel analysis of the Harderian gland transcriptome response to Newcastle disease virus in two inbred chicken lines

Behind each eye of the chicken resides a unique lymph tissue, the Harderian gland, for which RNA sequencing (RNA-seq) analysis is novel. We characterized the response of this tissue to Newcastle disease virus (NDV) in two inbred lines with different susceptibility to NDV across three time points. Three-week-old relatively resistant (Fayoumi) and relatively susceptible (Leghorn) birds were inoculated with a high-titered (107EID50) La Sota strain of NDV via an oculonasal route. At 2, 6, and 10 days post infection (dpi) Harderian glands were collected and analyzed via RNA-seq. The Fayoumi had significantly more detectable viral transcripts in the Harderian gland at 2 dpi than the Leghorn, but cleared the virus by 6 dpi. At all three time points, few genes were declared differentially expressed (DE) between the challenged and nonchallenged birds, except for the Leghorns at 6 dpi, and these DE genes were predicted to activate an adaptive immune response. Relative to the Leghorn, the Fayoumi was predicted to activate more immune pathways in both challenged and nonchallenged birds suggesting a more elevated immune system in the Fayoumis under homeostatic conditions. Overall, this study helped characterize the function of this important tissue and its response to NDV.

Avian Encephalomyelitis in Layer Pullets Associated with Vaccination

SUMMARY Avian encephalomyelitis (AE) was diagnosed in three flocks of leghorn layer pullets following AE vaccination. Ages of the birds were 11, 12, and 14 wk. The submissions came from three different companies located in two geographic areas of the Central Valley of California. The clinical signs included birds down on their legs, unilateral recumbency or sitting on their hocks, lethargy, reluctance to move, dehydration, unevenness in size, low weight, tremors of the head in a few birds, and mildly to moderately elevated mortality. The flocks had been vaccinated against fowl pox and AE with a combined product in the wing-web 2 wk prior to the onset of AE clinical signs. Histopathologic examination revealed lesions consistent with AE, including lymphocytic perivascular infiltration and neuronal central chromatolysis in the brain and spinal cord, as well as gliosis in the cerebellar molecular layer. The AE virus was detected by reverse-transcriptase PCR in the brain homogenate from three cases and peripheral nerves in one case. Additionally, the AE virus was isolated in specific-pathogen-free (SPF) embryonated eggs from brain tissue pool samples. Other avian viral infections capable of causing encephalitis, including avian paramyxoviruses, avian influenza virus (AIV), West Nile virus (WNV), eastern equine encephalitis virus (EEEV), and western equine encephalitis virus (WEEV), were ruled out by attempting virus isolation and molecular procedures.

Biosecurity Assessment and Seroprevalence of Respiratory Diseases in Backyard Poultry Flocks Located Close to and Far from Commercial Premises

SUMMARY Raising backyard chickens is an ever-growing hobby in the United States. These flocks can be a substrate for respiratory disease amplification and transmission to commercial facilities. Five hundred fifty-four chickens from 41 backyard flocks were sampled in this study. ELISA kits were used to detect antibodies against avian influenza (AI), infectious laryngotracheitis (ILT), Newcastle disease (ND), infectious bronchitis (IB), Ornithobacterium rhinotracheale (ORT), Mycoplasma gallisepticum (MG), and Mycoplasma synoviae (MS). All visited flock owners answered a biosecurity questionnaire that assessed biosecurity measures. The questionnaire revealed that backyard poultry owners lack simple biosecurity measures such as use of dedicated shoes, their chicken sources are unreliable, and few of them benefit from veterinary oversight. Only one flock had a clear vaccination history against ND and IB. ORT, ND, IB, MS, MG, and ILT were the most seroprevalent in backyard poultry flocks with 97% (41/42), 77.5% (31/40), 75% (30/40), 73% (31/42), 69% (29/42), and 45% (19/42), respectively. The vaccinated flock was not considered in these calculations. When examining the distance between backyard flocks and the nearest commercial poultry facility, ND and MG were significantly more likely to be found in backyard flocks close to (<4 miles) whereas ORT was significantly more likely in backyard chickens located far from (>4 miles) commercial poultry. Birds purchased directly from National Poultry Improvement Plan hatcheries showed a reduced ND, MG, and MS antibody prevalence. Wearing dedicated shoes decreased MS antibody-positive birds. Finally, history of wild bird contact had a clear effect on an increased seroprevalence of NDV and MG. Serological results suggest that backyard poultry flocks have the potential to serve as a reservoir or amplifier for poultry respiratory diseases. The information generated in this project should direct extension efforts toward emphasizing the importance of small flock biosecurity and chick acquisition sources.

Evolution of avian encephalomyelitis virus during embryo-adaptation

Wild-type avian encephalomyelitis virus (AEV) causes neurological signs in young chicks but no disease in pullets after oral or intracutaneous infection. However, if the virus gets embryo-adapted by serial passaging in chicken embryos, it will cause AE after intracutaneous infection in chickens of all ages. Recently, several cases of AE in layer pullets occurring shortly after intracutaneous vaccination were described. The present investigation was initiated to determine if vaccines that had inadvertently been embryo-adapted were responsible for these outbreaks. Virus isolation was done from two vaccines and one field sample. One of the vaccines had been used in one of the flocks before the outbreak. After the first passage, regardless of the inoculum, no embryo was paralyzed, indicating that the vaccines and the field isolate were not embryo-adapted. After seven passages all three strains were fully embryo-adapted causing typical lesions in the embryos. Viral load as determined by RT-qPCR remained constant during the passages. Partial sequences of the VP2 gene of vaccines, the field sample and four other field isolates were nearly identical and highly similar to published sequences from all over the world; only sequences originating from non-vaccinated birds were clearly set apart. Analysis of whole genomes identified two single nucleotide polymorphisms (SNPs) that distinguished wild-type and embryo-adapted strains. Sanger sequencing brains and nerves of the five field isolates and of the first, third and fifth passages of the isolates showed that the mutations indicating embryo-adaptation were first observed in the fifth passage.